Methods of simultaneously treating mucositis and fungal infection

ABSTRACT

A method for simultaneously treating mucositis and fungal infection in a mammal in need thereof, said method comprising administering to said mammal an effective amount of an anti-mucositis and anti-fungal pharmaceutical composition consisting of a tetracycline compound in an amount that is effective to simultaneously treat mucositis and fungal infection, but has substantially no antibiotic activity.

The present application claims benefit of U.S. provisional applicationSer. No. 60/377,998, filed May 6, 2002, which is incorporated herein byreference.

BACKGROUND OF INVENTION

Mucositis is a disease characterized by inflammation of the mucosa anddestruction of the mucosa epithelium. Such destruction results inerythema, ulcerations and severe pain.

Mucositis often arises in mammals that have compromised immune systems.For example, mucositis often appears as a complication of antineoplastictherapy, such as cancer chemotherapy and/or radiation therapy.

Fungal growth is also seen in patients whose immune systems have beencompromised, such as AIDS patients or chemotherapy patients. Fungalgrowth often accompanies mucositis.

Methods for treating mucositis have been disclosed. For example, Soniset al. have disclosed the use of inflammatory cytokine inhibitors, MMPinhibitors and/or mast cell inhibitors to treat mucositis.(International PCT application WO 99/45910.) Examples of MMP inhibitorsare said to include tetracyclines, such as minocycline, tetracyclineHCl, and doxycycline. Sonis et al. state that it is preferred to includean “antimicrobial agent” in their treatment. The only reason given bySonis et al. for adding an antimicrobial agent is that the presence ofbacteria leads to secondary infections and amplified tissue damage.Sonis et al. neither mention, nor suggest, including anti-fungal agents.

Lawter et al. acknowledge the disclosure by Sonis et al. of the use ofMMP inhibitors to treat mucositis. (International PCT application WO01/19362.) According to Lawter et al., the only MMP inhibitors whichappear to significantly reduce the symptoms of the mucositis are thetetracyclines. They attempt to reduce side effects by using atetracycline that is poorly absorbed from the gastro-intestinal tract. Atetracycline is defined as being poorly absorbed from thegastrointestinal tract if it has a bioavailability of about 10% or less.Lawter et al. describe fungi as not being susceptible to tetracyclines.Accordingly, Lawter et al. disclose that their formulation mayoptionally contain an anti-fungal agent.

Antibiotics, such as tetracyclines, have long been consideredineffective as anti-fungal agents. (Lu et al., Journal of DentalResearch, AADR Abstracts, 80:141, No. 845, (January 2001).)Nevertheless, Lu et al. tested the effects of two chemically modifiednon-antibiotic tetracyclines,6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline (CMT-3) and6-α-deoxy-5-hydroxy-4-de(dimethylamino)tetracycline (CMT-8), in vitroagainst eleven different species of fungi. CMT-3 showed anti-fungalactivity with eight of the eleven species. However, CMT-8 was said toshow weak or no anti-fungal activity.

Most current anti-fungal agents have significant toxic side effects.Therefore, the possibility of using tetracyclines as anti-fungal agentsappears attractive. Clearly, however, the state of the art teachingsregarding the clinical efficacy of tetracyclines as anti-fugal agents,as described above, is contradictory.

As stated above, many patients, such as patients with compromised immunesystems, are susceptible to both mucositis and fungal infections.Accordingly, there is a need for a method of simultaneously treating apatient suffering from both types of infections. It is especiallyadvantageous if a single agent would be effective to treat both types ofinfections. The use of a single agent would reduce both the cost andside effects of treatment.

SUMMARY OF THE INVENTION

The present invention provides a method for simultaneously treatingmucositis and fungal infection in a mammal in need thereof. The methodcomprises administering to the mammal an effective amount of ananti-mucositis and anti-fungal pharmaceutical composition consisting ofa tetracycline compound in an amount that is effective to simultaneouslytreat mucositis and fungal infection, but has substantially noantibiotic activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the photoirritancy factor (PIF) for some tetracyclinecompounds. For structure K, the compounds indicated are as follows: COLR7 R8 R9 308 hydrogen hydrogen amino 311 hydrogen hydrogen palmitamide306 hydrogen hydrogen dimethylamino

For structures L, M, N or 0 the compounds indicated are as follows: COLR7 R8 R9 801 hydrogen hydrogen acetamido 802 hydrogen hydrogendimethylaminoacetamido 804 hydrogen hydrogen nitro 805 hydrogen hydrogenaminoFor structure P, R8 is hydrogen and R9 is nitro (COL-1002).

FIG. 2 shows a Sample Dose Response Curve of the Positive ControlChlorpromazine for use in PIF calculations.

FIG. 3 shows a Sample Dose Response Curve for use in MPE calculations.

DETAILED DESCRIPTION OF INVENTION

The present invention provides methods of simultaneously treatingmucositis and fungal infection in a mammal.

Mucositis, as defined herein, includes any inflammation of the mucosa.The mucosa refers to the epithelial tissue that lines the internalcavities of the body. For example, the mucosa comprises the alimentarycanal, including the mouth, esophagus, stomach, intestines, and anus;the respiratory tract, including the nasal passages, trachea, bronchi,and lungs; and the genitalia.

A fungal infection as defined herein includes any infection caused byfungi. Fungi include any eukaryotic single celled organism characterizedby the absence of chlorophyll and by the presence of a rigid cell wall.The fungi of interest in the present specification are clinicallysignificant fungi, i.e. fungi which grow in or on mammals. Examples ofclinically significant fungi include Cryptococcus species, Canididaalbicans, Rhizopus species, Aspergillus fumigatus, Penicillium species,Absidia species, Scedosporium apiospermum, Phialophora verrucosa,Cunninghamella species, Tricothecium species, Ulocladium species, andFonsecae species.

The method of simultaneously treating mucositis and fungal infectioncomprises the administration of an anti-mucositis and anti-fungalpharmaceutical composition consisting of a tetracycline compound. Thetetracycline compound is administered in an amount which is effective tosimultaneously treat mucositis and a fungal infection, but which hassubstantially no antibiotic activity.

The tetracyclines are a class of compounds of which tetracycline is theparent compound. The tetracycline compounds include theirpharmaceutically acceptable salts. Tetracycline has the followingstructure:

The numbering system of the multiple ring nucleus is as follows:

Tetracycline, as well as the 5-OH (oxytetracycline, e.g. Terramycin) and7-Cl (chlorotetracycline, e.g. Aureomycin) derivatives, exist in nature,and are all well known antibiotic compounds. Semisynthetic derivativessuch as 7-dimethylaminotetracycline (minocycline) and6α-deoxy-5-hydroxytetracycline (doxycycline) are also known tetracyclineantibiotic compounds.

Some examples of antibiotic tetracycline compounds include doxycycline,minocycline, tetracycline, oxytetracycline, chlortetracycline,demeclocycline, lymecycline, and sancycline. Doxycycline is preferablyadministered as its hyclate salt or as a hydrate, preferablymonohydrate.

Non-antibiotic tetracycline compounds are structurally related to theantibiotic tetracyclines, but have had their antibiotic activitysubstantially or completely eliminated by chemical modification, asdiscussed in more detail below. For example, non-antibiotic tetracyclinecompounds are incapable of achieving antibiotic activity comparable tothat of doxycline unless the concentration of the non-antibiotictetracycline is at least about ten times, preferably at least abouttwenty five times, greater than that of doxycycline.

Examples of chemically modified non-antibiotic tetracyclines (CMT's)include, 4-de(dimethylamino)tetracycline (CMT-1), tetracyclinonitrile(CMT-2), 6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline (CMT-3),7-chloro-4-de(dimethylamino)tetracycline (CMT-4), tetracycline pyrazole(CMT-5), 4-hydroxy-4-de(dimethylamino)tetracycline (CMT-6),4-de(dimethylamino)-12α-deoxytetracycline (CMT-7),6-deoxy-5α-hydroxy-4-de(dimethylamino)tetracycline (CMT-8),4-de(dimethylamino)-12α-deoxyanhydrotetracycline (CMT-9),4-de(dimethylamino)minocycline (CMT-10). (COL and CMT are usedinterchangeably throughout this specification.)

Tetracycline derivatives, for purposes of the invention, may be anytetracycline derivative, including those compounds disclosed genericallyor specifically in U.S. patent application Ser. No. 09/573,653, filed onMay 18, 2000; International Application No. PCT/US01/16272 filed on May18, 2001; and U.S. patent application Ser. No. 10/274,841, filed Oct.18, 2002, which are herein incorporated by reference. Some examples ofchemically modified non-antibiotic tetracyclines include Structures C-Z.(See Index of Structures.)

The tetracycline compounds can be in the form of pharmaceuticallyacceptable salts of the compounds. Pharmaceutically acceptable salts maybe prepared from the corresponding tetracycline compounds and an acid orbase. The acids may be inorganic or organic acids. Examples of inorganicacids include hydrochloric, hydrobromic, nitric hydroiodic, sulfuric,and phosphoric acids. Examples of organic acids include carboxylic andsulfonic acids. The organic acids may be aliphatic, aromatic,aliphatic-aromatic or aromatic-aliphatic. Some examples of organic acidsinclude formic, acetic, phenylacetic, propionic, succinic, glycolic,glucuronic, maleic, furoic, glutamic, benzoic, toluic, anthranilic,salicylic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic,panthenoic, benzenesulfonic, stearic, sulfanilic, alginic, tartaric,citric, gluconic, gulonic, arylsulfonic, and galacturonic acids.Appropriate organic bases may be selected, for example, fromN,N-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,ethylenediamine, meglumine (N-methylglucamine), and procaine.

The tetracycline compound is administered in an amount that is effectiveto simultaneously treat mucositis and fungal infection, but hassubstantially no antibiotic activity. A treatment is effective if itcauses a reduction or inhibition of the symptoms associated withmucositis and fungal infection.

The minimal effective amount of the tetracycline compound administeredto a mammal is the lowest amount capable of providing effectivesimultaneous treatment of mucositis and fungal infection. Some examplesof minimal amounts include 10%, 20%, 30% and 40% of an antibioticamount.

The maximal effective amount of the tetracycline compound administeredto a mammal is the highest amount that does not significantly preventthe growth of microbes, e.g. bacteria. Some examples of maximal amountsinclude 50%, 60%, 70% and 80% of an antibiotic amount.

The amount of a tetracycline compound which is administered can bemeasured by daily dose and by serum level.

Tetracycline compounds that have significant antibiotic activity may,for example, be administered in a dose which is 10-80% of the antibioticdose. More preferably, the antibiotic tetracycline compound isadministered in a dose which is 40-70% of the antibiotic dose.

Antibiotic daily doses are known in art. Some examples of antibioticdoses of members of the tetracycline family include 50, 75, and 100mg/day of doxycycline; 50, 75, 100, and 200 mg/day of minocycline; 250mg of tetracycline one, two, three, or four times a day; 1000 mg/day ofoxytetracycline; 600 mg/day of demeclocycline; and 600 mg/day oflymecycline.

Examples of the maximum non-antibiotic doses of tetracyclines based onsteady-state pharmacokinetics are as follows: 20 mg/twice a day fordoxycycline; 38 mg of minocycline one, two, three or four times a day;and 60 mg of tetracycline one, two, three or four times a day.

In a preferred embodiment, doxycycline is administered in a daily amountof from about 30 to about 60 milligrams, but maintains a concentrationin human plasma below the threshold for a significant antibiotic effect.

In an especially preferred embodiment, doxycycline hyclate isadministered at a 20 milligram dose twice daily. Such a formulation issold for the treatment of periodontal disease by CollaGenexPharmaceuticals, Inc. of Newtown, Pa. under the trademark Periostat®.

The administered amount of a tetracycline compound described by serumlevels follows.

An antibiotic tetracycline compound is advantageously administered in anamount that results in a serum tetracycline concentration which is10-80%, preferably 40-70%, of the minimum antibiotic serumconcentration. The minimum antibiotic serum concentration is the lowestconcentration known to exert a significant antibiotic effect.

Some examples of the approximate antibiotic serum concentrations ofmembers of the tetracycline family follow. A single dose of two 100 mgminocycline HCl tablets administered to adult humans results inminocycline serum levels ranging from 0.74 to 4.45 μg/ml over a periodof an hour. The average level is 2.24 μg/ml.

Two hundred and fifty milligrams of tetracycline HCl administered everysix hours over a twenty-four hour period produces a peak plasmaconcentration of approximately 3 μg/ml. Five hundred milligrams oftetracycline HCl administered every six hours over a twenty-four hourperiod produces a serum concentration level of 4 to 5 μg/ml.

In one embodiment, the tetracycline compound can be administered in anamount which results in a serum concentration between about 0.1 and 10.0μg/ml, more preferably between 0.3 and 5.0 μg/ml. For example,doxycycline is administered in an amount which results in a serumconcentration between about 0.1 and 0.8 μg/ml, more preferably between0.4 and 0.7 μg/ml.

Some examples of the plasma antibiotic threshold levels of tetracyclinesbased on steady-state pharmacokinetics are as follows: 1.0 μg/ml fordoxycycline; 0.8 μg/ml for minocycline; and 0.5 μg/ml for tetracycline.

Non-antibiotic tetracycline compounds can be used in higher amounts thanantibiotic tetracyclines, while avoiding the indiscriminate killing ofmicrobes, and the risk of emergence of resistant microbes. For example,6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline (CMT-3) may beadministered in doses of about 40 to about 200 mg/day, or in amountsthat result in serum levels of about 1.55 μg/ml to about 10 μg/ml.

The actual preferred amounts of tetracycline compounds in a specifiedcase will vary according to the particular compositions formulated, themode of application, the particular sites of application, and thesubject being treated (e.g. age, gender, size, tolerance to drug, etc.)

Preferably, the tetracycline compounds have low phototoxicity, or areadministered in an amount that results in a serum level at which thephototoxicity is acceptable. Phototoxicity is a chemically-inducedphotosensitivity that occurs upon exposure to light, in particularultraviolet light. Such photosensitivity renders skin susceptible todamage, e.g. sunburn, blisters, accelerated aging, erythemas andeczematoid lesions. The preferred amount of the tetracycline compoundproduces no more phototoxicity than is produced by the administration ofa 40 mg total daily dose of doxycycline.

There are several methods by which to quantify phototoxicity. One methodis called photoirritancy factor (PIF). The PIF is the ratio of an IC₅₀value in the absence of light to an IC₅₀ value in the presence of light.

In calculating PIF values, the data resulting from the assay procedurecan be interpreted by different methods. For example, during the periodMar. 2, 1999 to Apr. 16, 1999, PIF values were obtained using thephototoxicity software and its curve-fitting algorithms available at thetime. In the present specification, this earlier phototoxicitycalculation is referred to as PIF 1. At a PIF1 value of 1, a compound isconsidered to have no measurable phototoxicity. A PIF1 value greaterthan 5 is indicative of phototoxic potential of a compound.

As explained in more detail in Example 37 below, 3T3 phototoxicity assayhas undergone extensive validation since April 1999, and has now beenincorporated into a draft guideline by the Organization of EconomicCooperation and Development (OECD) (Draft Guideline 432). In the presentspecification, this revised phototoxicity calculation is referred to asPIF2. A PIF2 value of less than 2 is considered non-phototoxic, 2 toless than 5 is considered potentially phototoxic, and 5 or greater isconsidered clearly phototoxic.

PIF2 values are more refined than the PIF1 values. Qualitatively thedifferences between the PIF1 and PIF2 values are not significant. Forexample, the mean PIF1 values for COL 10 and COL 1002 are 1.82 and 1.0,respectively. The mean PIF2 values of COL 10 and COL 1002 are 2.04 and1.35, respectively.

As explained in the Examples section, PIF values cannot be determinedfor many compounds. Another method by which to quantify relativephototoxicity is called mean photo effect (MPE). MPE values can bedetermined for compounds in virtually all cases. Thus, MPE values aremore consistent and reliable than PFE values.

The MPE is a measure of the difference between the cytotoxicity inducedby the test chemical in the presence and absence of light. It comparesthe responses over the range of doses selected using the twodose-response curves produced from the boot-strap analysis of theindividual data points (Holzhütter 1995 and 1997). An example isprovided in FIG. 3 (Peters and Holzhütter (2002)). This method ofanalysis is particularly suited to cases where the IC₅₀ value cannot becalculated for one or both concentration response curves.

MPE values of <0.1 (including negative values) are considered indicativeof a nonphototoxin, values of 0.1 to <0.15 are considered probablephototoxins, and values greater than and equal to 0.15 are considered tobe clear phototoxins.

A class of low phototoxicity tetracyline derivatives has less thanapproximately 75% of the phototoxicity of minocycline, preferably lessthan approximately 70%, more preferably less than approximately 60%, andmost preferably less than approximately 50%. Minocycline has a PIF1 ofabout 2.04, and an MPE of about 0.041.

The class of low phototoxicity tetracycline compound derivativesincludes those derivatives having PIF 1 or PIF 2 values of approximately1, i.e. 1 to about 2, preferably 1 to about 1.5. The class of lowphototoxicity tetracycline derivatives optimally have MPE values of lessthan 0.1. Members of this class include, but are not limited to,tetracycline compounds having general formulae:

Structure K

wherein: R7, R8, and R9 taken together in each case, have the followingmeanings: R7 R8 R9 hydrogen hydrogen amino hydrogen hydrogen palmitamidehydrogen hydrogen dimethylamino trimethylammonium hydrogen hydrogen andSTRUCTURE L STRUCTURE M STRUCTURE N STRUCTURE O

wherein: R7, R8, and R9 taken together in each case, have the followingmeanings: R7 R8 R9 hydrogen hydrogen acetamido hydrogen hydrogendimethylaminoacetamido hydrogen hydrogen nitro hydrogen hydrogen aminoand STRUCTURE Pwherein: R8 and R9 taken together are, respectively, hydrogen and nitro.

The tetracycline compounds are preferably administered systemically ortopically. For the purposes of this specification, “systemicadministration” means administration to a human by a method that causesthe compounds to be absorbed into the bloodstream.

For example, the tetracycline compounds can be administered orally byany method known in the art. For example, oral administration can be bytablets, capsules, pills, troches, elixirs, suspensions, syrups, wafers,chewing gum and the like.

Additionally, the tetracycline compounds can be administered enterallyor parenterally, e.g., intravenously, intramuscularly, orsubcutaneously, as injectable solutions or suspensions;intraperitoneally; or rectally. Administration can also be intranasally,in the form of, for example, an intranasal spray; or transdermally, inthe form of, for example, a patch.

For the pharmaceutical purposes described above, the tetracyclinecompounds 5 can be formulated in pharmaceutical preparations optionallywith a suitable pharmaceutical carrier (vehicle) or excipient asunderstood by practitioners in the art. These preparations can be madeaccording to conventional chemical methods.

In the case of tablets and capsules for oral use, carriers which arecommonly used include lactose and corn starch. Lubricating agents suchas magnesium stearate are commonly added. Further examples of carriersand excipients include milk, sugar, certain types of clay, gelatin,stearic acid or salts thereof, calcium stearate, talc, vegetable fats oroils, gums and glycols.

When aqueous suspensions are used for oral administration, emulsifyingand/or suspending agents are commonly added. In addition, sweeteningand/or flavoring agents may be added to the oral compositions.

For intramuscular, intraperitoneal, subcutaneous and intravenous use,sterile solutions of the tetracycline compounds can be employed. The pHof the solutions are preferably adjusted and buffered. For intravenoususe, the total concentration of the solute(s) can be controlled in orderto render the preparation isotonic.

The tetracycline compounds of the present invention optionally furthercomprise one or more additional pharmaceutically acceptableingredient(s) such as alum, stabilizers, buffers, coloring agents,flavoring agents, and the like.

The tetracycline compound may be administered intermittently. Forexample, the tetracycline compound may be administered 1-6 times a day,preferably 1-4 times a day.

Alternatively, the tetracycline compound may be administered bysustained release. Sustained release administration is a method of drugdelivery to achieve a certain level of the drug over a particular periodof time. The level typically is measured by serum concentration. Furtherdescription of methods of delivering tetracycline compounds by sustainedrelease can be found in the patent application, “Controlled Delivery ofTetracycline and Tetracycline Derivatives,” filed on Apr. 5, 2001 andassigned to CollaGenex Pharmaceuticals, Inc. of Newtown, Pa. Theaforementioned application is incorporated herein by reference in itsentirety. For example, 40 milligrams of doxycycline may be administeredby sustained release over a 24 hour period.

For topical application, the tetracycline compounds are placed incarrier compositions deemed to be suited for topical use, such as gels,salves, lotions, creams, ointments and the like. The carriercompositions can also be incorporated into a support base or matrixwhich can be directly applied to the mucosa. Examples of a support baseor matrix include gauze or bandages.

The carrier compositions can comprise a tetracycline compound in amountsof up to about 25% (w/w). Amounts of from about 0.1% to about 10% arepreferred.

Topical application is preferred for particular non-antibiotictetracycline compounds which have only limited biodistribution, e.g.CMT-5.

Combined or coordinated topical and systemic administration of thetetracycline compounds is also contemplated under the invention. Forexample, a systemically non-absorbable non-antibiotic tetracyclinecompound can be administered topically, while a tetracycline compoundcapable of substantial absorption and effective systemic distribution ina human can be administered systemically.

The tetracycline compounds are prepared by methods known in the art. Forexample, natural tetracyclines may be modified without losing theirantibiotic properties, although certain elements of the structure mustbe retained. The modifications that may and may not be made to the basictetracycline structure have been reviewed by Mitscher in The Chemistryof Tetracyclines, Chapter 6, Marcel Dekker, Publishers, New York (1978).According to Mitscher, the substituents at positions 5-9 of thetetracycline ring system may be modified without the complete loss ofantibiotic properties. Changes to the basic ring system or replacementof the substituents at positions 1-4 and 10-12, however, generally leadto synthetic tetracyclines with substantially less or effectively noantibiotic activity.

Further methods of preparing the tetracycline compounds are described inthe examples.

EXAMPLES

The following examples serve to provide further appreciation of theinvention but are not meant in any way to restrict the effective scopeof the invention.

Preparation of Compounds

Example 14-Dedimethylamino-7-dimethylamino-6-demethyl-6-deoxy-9-nitrotetracyclinesulfate

To a solution of one millimole of4-dedimethylamino-7-dimethylamino-6-demethyl-6-deoxytetracycline in 25ml of concentrated sulfuric acid at 0° C. was added 1.05 mmole ofpotassium nitrate. The resulting solution was stirred at ice bathtemperature for 15 minutes and poured in one liter of cold ether withstirring. The precipitated solid was allowed to settle and the majorityof solvent decanted. The remaining material was filtered through asintered glass funnel and the collected solid was washed well with coldether. The product was dried in a vacuum desiccator overnight.

Example 29-amino-dedimethylamino-7diethylaamino-6-demethyl-6-deoxytetracyclinesulfate

To a solution of 300 mg of the 9-nitro compound from example 1, in 30 mlof ethanol was added 50 mg of Pt0₂. The mixture was hydrogenated atatmospheric pressure until the theoretical amount of hydrogen wasabsorbed. The system is flushed with nitrogen, the catalyst PtO₂ isfiltered and the filtrate added dropwise to 300 ml of ether. The productthat separates is filtered and dried in a vacuum desiccator.

Example 39-Acetamido-4-dedimethylamino-7-dimethylamino-6-demethyl-6-deoxytetracyclinesulfate

To a well stirred cold solution of 500 mg of9-amino-4-dedimethylamino-7-dimethylamino-6-demethyl-6-deoxytetracyclinesulfate from example 2, in 2.0 ml of 1.3-dimethyl-2-imidizolidinone, 500mg of sodium bicarbonate was added followed by 0.21 ml of acetylchloride. The mixture is stirred at room temperature for 30 minutes,filtered and the filtrate was added dropwise to 500 ml of ether. Theproduct that separated was filtered and dried in a vacuum desiccator.

Example 44-Dedimethylamino-7-dimethylamino-6-demethyl-6-deoxy-9-diazoniumtetracyclinesulfate

To a solution of 0.5 g of9-amino-4-dedimethylamino-7-dimethylamino-6-demethyl-6-deoxytetracyclinesulfate, from example 2, in 10 ml of 0.1N hydrochloric acid in methanolcooled in an ice bath, 0.5 ml of n-butyl nitrite was added. The solutionwas stirred at ice bath temperature for 30 minutes and then poured into250 ml of ether. The product that separated was filtered, washed withether and dried in a vacuum desiccator.

Example 59-Azido-4-dedimethylamino-7-dimethylamino-6-demethyl-6-deoxytetracyclinesulfate

To a solution of 0.3 mmole of4-dedimethylamino-7-dimethylamino-6-demethyl-6-deoxy-9-diazoniumtetracyclinesulfate, from example 4, 10 ml of 0.1 N methanolic hydrogen chloride wasadded 0.33 mmole of sodium azide. The mixture was stirred at roomtemperature for 1.5 hours. The reaction mixture was then poured into 200ml of ether. The product that separated was filtered and dried in avacuum desiccator.

Example 69-Amino-8-chloro-4-dedimethylamino-7-dimethylamino-6-demethyl-6-deoxy-tetracyclinesulfate

One gram of9-azido-4-dedimethylamino-7-dimethylamino-6-demethyl-6-deoxytetracyclinehydrochloride, from example 4, was dissolved in 10 ml of concentratedsulfuric acid saturated with HCL at 0° C. The mixture was stirred at icebath temperature for 1.5 hours and then slowly added dropwise to 500 mlof cold ether. The product that separated was filtered, washed withether and dried in a vacuum desiccator.

Example 74-Dedimethylamino-7-dimethylamino-6-demethyl-6-deoxy-9-ethoxythiocarbonylthio-tetracyclinesulfate

A solution of 1.0 mmole of4-dedimethylamino-7-dimethylamino-6-demethyl-6-deoxy-9-diazoniumtetracyclinesulfate, from example 4, in 15 ml of water was added to a solution of1.15 mmole of potassium ethyl xanthate in 15 ml of water. The mixturewas stirred at room temperature for one hour. The product separated andwas filtered and dried in a vacuum desiccator.

Example 8A General Procedure for Nitration

To 1 mmole of a 4-dedimethylamino-6-deoxytetracycline in 25 ml ofconcentrated sulfuric acid at 0° C. was added 1 mmole of potassiumnitrate with stiring. The reaction solution was stirred for 15 minutesand then poured into 100 g of chopped ice. The aqueous solution wasextracted 5 times with 20 ml of butanol each time. The butanol extractswere washed three times with 10 ml of water each time, and concentratedin vacuo to a volume of 25 ml. The light yellow crystalline solid whichprecipitated was filtered, washed with 2 ml of butanol and dried invacuo at 60° C. for 2 hours. This solid was a mixture of the twomononitro isomers.

Example 8B 4-Dedimethylamino-6-deoxy-9-nitrotetracycline

To 980 mg of the nitration product from4-dedimethylamino-6-deoxytetracycline (a mixture of the 2 isomers) in 25ml of methanol was added enough triethylamine to dissolve the solid. Thefiltered solution (pH 9.0) was adjusted to pH 5.2 with concentratedsulfuric acid. A crystalline yellow solid (236 mg.) was obtained (29%yield). The material at this point was quite pure and contained onlysmall amounts of the 7-isomer. Final purification was accomplished byliquid partition chromatography using a diatomaceous earth packed columnand the solvent system: chloroform: butanol: 0.5 M phosphate buffer (pH2) (16:1:10).

Example 9 4-Dedimethylamino-6-deoxy-7-nitrotetracycline

The methanol filtrate from example 8 was immediately adjusted to pH 1.0with concentrated sulfuric acid. The light yellow crystalline solid,which was obtained as the sulfate salt. A purified free base wasobtained by adjusting an aqueous solution of the sulfate salt (25 mg/ml)to pH 5.2 with 2 N sodium carbonate.

Example 10 9-Amino-4-dedimethylamino-6-deoxytetracycline

To a solution of 300 mg of the 9-nitro compound, prepared in example 8,in 30 ml of ethanol was added 50 mg of PtO₂. The mixture washydrogenated at atmospheric pressure until the theoretical amount ofhydrogen was absorbed. The system is flushed with nitrogen, the PtO2catalyst is filtered and the filtrate added dropwise to 300 ml of ether.The solid that separates is filtered and dried in a vacuum desiccator.

Example 11 9-Acetamido-4-dedimethylamino-6-deoxytetracycline sulfate

To well stirred cold solution of 500 mg of9-amino4-dedimethylamino-6-deoxytetracycline sulfate, from example 10,in 2.0 ml of 1,3-dimethyl-2-imidazolidinone was added 500 mg of sodiumbicarbonate followed by 0.21 ml of acetyl chloride. The mixture wasstirred at room temperature for 30 minutes, filtered and the filtratewas added dropwise to 500 ml of ether. The solid that separated wasfiltered and dried in a vacuum desiccator.

Example 12 4-Dedimethylamino-6-deoxy-9-diazoniumtetracycline sulfate

To a solution of 0.5 g of 9-amino4-dedimethylamino-6-deoxytetracyclinesulfate, from example 10, in 10 ml of 0.1N hydrochloric acid in methanolcooled in an ice bath was added 0.5 ml of n-butyl nitrite. The solutionwas stirred at ice bath temperature for 30 minutes and the poured into250 ml of ether. The solid that separated was filtered, washed withether and dried in a vacuum desiccator.

Example 13 9-Azido4-dedimethylamino-6-deoxytetracycline sulfate

To a solution of 0.3 mmole of4-dedimethylamino-6-deoxy-9-diazoniumtetracycline sulfate, of example12, 10 ml of 0.1 N methanolic hydrogen chloride was added 0.33 mmole ofsodium azide. The mixture was stirred at room temperature for 1.5 hours.The reaction mixture was then poured into 200 ml of ether. The solidthat separated was filtered and dried in a vacuum desiccator.

Example 14 9-Amino-8-chloro4-dedimethylamino-6-deoxytetracycline sulfate

One gram of9-azido-4-dedimethylamino-7-dimethylamino-6-deoxytetracyclinehydrochloride, from example 13, was dissolved in 10 ml of concentratedsulfuric acid saturated with HCL at 0° C. The mixture was stirred at icebath temperature for 1.5 hours and then slowly added dropwise to 500 mlof cold ether. The solid that separated was filtered, washed and etherand dried in a vacuum desiccator.

Example 154-Dedimethylamino-6-deoxy-9-ethoxythiocarbonylthiotetracycline sulfate

A solution of 1.0 mmole of4-dedimethylamino-6-deoxy-9-diazoniumtetracycline sulfate, from example12, in 15 ml of water was added to a solution of 1.15 mmole of potassiumethyl xanthate in 15 ml of water. The mixture was stirred at roomtemperature for one hour. The solid that separated was filtered anddried in a vacuum desiccator.

Example 16 9-Dimethylamino-4-dedimethylamino-6-deoxytetracycline sulfate

To a solution of 100 mg. of the 9-amino compound from example 10, in 10ml of ethylene glycol monomethyl ether is added 0.05 ml of concentratedsulfuric acid, 0.4 ml. of a 40% aqueous formaldehyde solution and 100 mgof a 10% palladium on carbon catalyst. The mixture is hydrogenated underatmospheric pressure and room temperature for 20 minutes. The catalystwas filtered and the filtrate was evaporated to dryness under reducedpressure. The residue is dissolved in 5 ml of methanol and this solutionwas added to 100 ml of ether. The product that separated was filteredand dried, yield, 98 mg.

Example 17 7-Amino-4-dedimethylamino-6-deoxytetracycline

This compound can be made using Procedure A or B. Procedure A. To asolution of 300 mg of the 7-nitro compound, from example 1, in 30 ml ofethanol was added 50 mg of PtO₂. The mixture was hydrogenated atatmospheric pressure until the theoretical amount of hydrogen wasabsorbed. The system is flushed with nitrogen, the catalyst PtO₂ isfiltered and the filtrate added dropwise to 300 ml of ether. The solidthat separates is filtered and dried in a vacuum desiccator.

Procedure B. 1 g of 6-deoxy4-dedimethylamino-tetracycline was dissolvedin 7.6 ml THF and 10.4 ml methanesulfonic acid at −10° C. After warmingthe mixture to 0° C. a solution of 0.86 g of dibenzyl azodicarboxylatewas added and the mixture stirred for 2 hours at 0° C. to yield7-[1,2-bis(carbobenzyloxy)hydrazino]-4-dedimethylamino-6-deoxytetracycline.A solution of 1 millimole of this material in 70 ml 2-methoxyethanol,and 300 mg 10% Pd-C was hydrogenated at room temperature to give7-amino-6-deoxy-4-dedimethylaminotetracycline.

Example 18 7-Amino-6-deoxy-5-hydroxy-4-dedimethylaminotetracycline

1 g of 6-deoxy-5-hydroxy-4-dedimethylaminotetracycline 3 was dissolvedin 7.6 ml THF and 10.4 ml methanesulfonic acid at −10° C. After warmingthe mixture to 0° C. a solution of 0.86 g dibenzyl azodicarboxylate in0.5 ml THF was added and the mixture stirred for 2 hours at 0° C. toyield7-[1,2-bis(carbobenzyloxy)hydrazino]-4-dedimethylamino-6-deoxy-5-hydroxytetracycline.A solution of 1 millimole of this material in 70 ml 2-methoxyethanol,and 300 mg 10% Pd-C was hydrogenated at room temperature to give7-amino-6-deoxy-5-hydroxytetracycline.

Example 19 7-Acetamido-4-dedimethylamino-6-deoxy-5-hydroxytetracyclinesulfate.

To well stirred cold solution of 500 mg of7-amino-4-dedimethylamino-6-deoxy-5-hydroxytetracycline sulfate, fromexample 18, in 2.0 ml of 1,3-dimethyl-2-imidazolidinone was added 500 mgof sodium bicarbonate followed by 0.21 ml of acetyl chloride. Themixture was stirred at room temperature for 30 minutes, filtered and thefiltrate was added dropwise to 500 ml of ether. The solid that separatedwas filtered and dried in a vacuum desiccator.

Example 20 4-Dedimethylamino-6-deoxy-5-hydroxy-7-diazoniumtetracyclinehydrochloride

To a solution of 0.5 g of7-amino-4-dedimethylamino-6-deoxy-5-hydroxytetracycline sulfate, fromexample 20, in 10 ml of 0.1N hydrochloric acid in methanol cooled in anice bath was added 0.5 ml of n-butyl nitrite. The solution was stirredat ice bath temperature for 30 minutes and then poured into 250 ml ofether. The solid that separated was filtered, washed with ether anddried in a vacuum desiccator.

Example 21 7-Azido-4-Dedimethylamino-6-deoxy-5-hydroxytetracycline

To a solution of 0.3 mmole of4-dedimethylamino-6-deoxy-5-hydroxy-7-diazoniumtetracyclinehydrochloride, from example 20, 10 ml of 0.1 N methanolic hydrogenchloride was added 0.33 mmole of sodium azide. The mixture was stirredat room temperature for 1.5 hours. The reaction mixture was then pouredinto 200 ml of ether. The solid that separated was filtered and dried ina vacuum desiccator.

Example 227-Amino-8-chloro-4-dedimethylamino-6-deoxy-5-hydroxytetracycline sulfate

One gram of7-azido-4-dedimethylamino-7-dimethylamino-6-deoxy-5-hydroxytetracyclinesulfate, from example 21, was dissolved in 10 ml of concentratedsulfuric acid (previously saturated with hydrogen chloride) at 0° C. Themixture was stirred at ice bath temperature for 1.5 hours and thenslowly added dropwise to 500 ml of cold ether. The solid that separatedwas filtered, washed with ether and dried in a vacuum desiccator.

Example 234-Dedimethylamino-6-deoxy-5-hydroxy-7-ethoxythiocarbonylthiotetracycline

A solution of 1.0 mmole of4-dedimethylamino-6-deoxy-5-hydroxy-7-diazoniumtetracyclinehydrochloride, from example 20, in 15 ml of water was added to asolution of 1.15 mmole of potassium ethyl xanthate in 15 ml of water.The mixture was stirred at room temperature for one hour. The solid thatseparated was filtered and dried in a vacuum desiccator.

Example 247-Dimethylamino-4-dedimethylamino-6-deoxy-5-hydroxytetracycline sulfate

To a solution of 100 mg of the 7-amino compound in 10 ml of ethyleneglycol monomethyl ether is added 0.05 ml of concentrated sulfuric acid,0.4 ml of a 40% aqueous formaldehyde solution and 100 mg of a 10%palladium on carbon catalyst. The mixture is reduced with hydrogen atatmospheric pressure and room temperature for 20 minutes. The catalystwas filtered and the filtrate was evaporated to dryness under reducedpressure. The residue is dissolved in 5 ml of methanol and this solutionwas added to 100 ml of ether. The product that separated was filteredand dried, yield, 78 mg.

Example 25 7-Diethylamino-4-dedimethylamino-5-hydroxytetracyclinesulfate

To a solution of 100 mg of the 7-amino compound in 10 ml of ethyleneglycol monomethyl ether is added 0.05 ml of concentrated sulfuric acid,0.4 ml of acetaldehyde and 100 mg of a 10% palladium on carbon catalyst.The mixture is reduced with hydrogen at atmospheric pressure at roomtemperature for 20 minutes. The catalyst was filtered and filtrate wasevaporated to dryness under reduced pressure. The residue is dissolvedin 5 ml of methanol and this solution was added to 100 ml of ether. Theproduct that separated was filtered and dried.

Example 26 4-Dedimethylamino-6-deoxy-7-diazoniumtetracyclinehydrochloride

To a solution of 0.5 g. of 7-amino-4-dedimethylamino-6-deoxytetracyclinesulfate, from example 17, in 10 ml of 0.1N hydrochloric acid in methanolcooled in an ice bath was added 0.5 ml of n-butyl nitrite. The solutionwas stirred at ice bath temperature for 30 minutes and then poured into250 ml of ether. The solid that separated was filtered, washed withether and dried in a vacuum desiccator.

Example 27 7-Azido-4-dedimethylamino-6-deoxytetracycline

To a solution of 0.3 mmole of4-dedimethylamino-6-deoxy-7-diazoniumtetracycline hydrochloride, fromexample 26, 10 ml of 0.1 N methanolic hydrogen chloride was added 0.33mmole of sodium azide. The mixture was stirred at room temperature for1.5 hours. The reaction mixture was then poured into 200 ml of ether.The solid that separated was filtered and dried in a vacuum desiccator.

Example 28 7-Amino-8-chloro-4-dedimethylamino-6-deoxytetracyclinesulfate

One gram of7-azido-4-dedimethylamino-7-dimethylamino-6-deoxytetracycline sulfatewas dissolved in 10 ml of concentrated sulfuric acid (previouslysaturated with hydrogen chloride) at 0° C. The mixture was stirred atice bath temperature for 1.5 hours and then slowly added dropwise to 500ml of cold ether. The solid that separated was filtered, washed withether and dried in a vacuum desiccator.

Example 294-Dedimethylamino-6-deoxy-7-ethoxythiocarbonylthiotetracycline

A solution of 1.0 mmole of4-dedimethylamino-6-deoxy-7-diazoniumtetracycline hydrochloride, fromexample 26, in 15 ml of water was added to a solution of 1.15 mmole ofpotassium ethyl xanthate in 15 ml of water. The mixture was stirred atroom temperature for one hour. The solid that separated was filtered anddried in a vacuum desiccator.

Example 30 7-Dimethylamino-4-dedimethylamino-6-deoxytetracycline sulfate

To a solution of 100 mg of the 7-amino compound, from example 26, in 10ml of ethylene glycol monomethyl ether is added 0.05 ml of concentratedsulfuric acid, 0.4 ml of a 40% aqueous formaldehyde solution and 100 mgof a 10% palladium on carbon catalyst. The mixture is reduced withhydrogen at atmospheric pressure and room temperature for 20 minutes.The catalyst was filtered and the filtrate was evaporated to drynessunder reduced pressure. The residue is dissolved in 5 ml of methanol andthis solution was added to 100 ml of ether. The product that separatedwas filtered and dried.

Example 319-Acetamido-8-chloro-4-dedimethylamino-7-dimethylamino-6-deoxy-6-demethyltetracycline

To well stirred cold solution of 500 mg of9-amino-8-chloro-4-dedimethylamino-6-deoxy-6-demethyl-7-dimethyl aminotetracycline sulfate, from example 6, in 2.0 ml of 1,3-dimethyl-2-imidazolidinone was added 500 mg of sodium bicarbonate followed by0.21 ml. of acetyl chloride. The mixture was stirred at room temperaturefor 30 minutes, filtered and the filtrate was added dropwise to 500 mlof ether. The solid that separated was filtered and dried in a vacuumdesiccator.

Example 328-Chloro4-dedimethylamino-7-dimethylamino-6-deoxy-6-demethyl-9-ethoxythiocarbonylthiotetracycline

A solution of 1.0 mmole of-8-chloro4-dedimethylamino-6-deoxy-6-demethyl-7-dimethylamino-9-diazoniumtetracycline hydrochloride in 15 ml of water was addedto a solution of 1.15 mmole of potassium ethyl xanthate in 15 ml ofwater. The mixture was stirred at room temperature for one hour. Thesolid that separated was filtered and dried in a vacuum desiccator.

Example 338-Chloro-9-dimethylamino-4-dedimethylamino-7-dimethylamino-6-deoxy-6-demethytetracyclinesulfate

To a solution of 100 mg. of the 9-amino compound, from example 6, in 10ml of ethylene glycol monomethyl ether is added 0.05 ml of concentratedsulfuric acid, 0.4 ml of acetaldehyde and 100 mg of a 10% palladium oncarbon catalyst. The mixture is reduced with hydrogen at atmosphericpressure and room temperature for 20 minutes. The catalyst was filteredand the filtrate was evaporated to dryness under reduced pressure. Theresidue is dissolved in 5 ml of methanol and this solution was added to100 ml of ether. The product that separated was filtered and dried.

Example 34N-(4-methylpiperazin-1-yl)methyl-4-dedimethylamino-6-demethyl-6-deoxytetracycline

An aqueous solution of 58 mg (37%) formaldehyde (0.72 mmol) was added toa solution of 203 mg (0.49 mmol) of4-dedimethylamino-6-demethyl-6-deoxytetracycline in 5.0 ml ethyleneglycol dimethyl ether. The mixture was stirred at room temperature for0.5 hours. 56 mg (0.56 mmol) of 1-methylpiperazine was then added andthe resulting mixture was stirred overnight and refluxed for 20 minutes.The mixture was then cooled and a solid product was collected byfiltration. The solid product was then washed with the solvent and driedby vacuum filtration.

Example 35N-(4-methylpiperazin-1-yl)methyl4-dedimethylamino-6-demethyl-6-deoxy-9-hexanoylaminotetracycline

An aqueous solution of 49 mg 37 % formaldehyde (0.60 mmol) was added toa solution of 146 mg (0.30 mmol) of4-dedimethylamino-6-demethyl-6-deoxy-9-hexanoylaminotetracycline in 5.0ml ethylene glycol dimethyl ether. The mixture was stirred at roomtemperature for 0.5 hours. 60 mg (0.60 mmol) of 1-methylpiperazine wasthen added and the resulting mixture was stirred overnight and refluxedfor 20 minutes. The mixture was then cooled and a solid product wascollected by filtration. The solid product was then washed with thesolvent and dried by vacuum filtration.

Example 364-Dedimethylamino-6-demethyl-6-deoxy-9-hexanoylaminotetracycline.

1.54 g (7.2 mmol) of hexanoic anhydride and 150 mg of 10% Pd/C catalystwere added to 300 mg (0.72 mmol) of4-dedimethylamino-6-demethyl-6-deoxytetracycline in 6.0 ml of1,4-dioxane and 6.0 ml of methanol. The mixture was hydrogenatedovernight at room temperature. The catalyst was removed by filtrationand the filtrate was concentrated under reduced pressure. The residuewas dissolved in 7 ml of ethyl acetate and trituated with 50 ml ofhexane to produce a solid product. The solid product was filtered anddried by vacuum filtration.

Example 37 Phototoxicity Determination

BALB/c 3T3 (CCL-163) cells were obtained from ATCC and cultured inantibiotic-free Dulbecco's Minimum Essential Medium (4.5 g/1glucose)(DMEM) supplemented with L-glutamine (4 mM and 10% newborn calfserum. The working cell bank was prepared and found to be free ofmycoplasma. Streptomycin sulfate (100 g/ml) and penicillin (100 IU/ml)were added to the medium after the cells were treated with test articlein 96-well plates.

Serial dilutions of the tetracycline derivatives were prepared in DMSOat concentrations 100× to final testing concentration. The COL dilutionsin DMSO were then diluted in Hanks' Balanced Salt Solution (HBSS) forapplication to the cells. The final DMSO concentration was 1% in treatedand control cultures. A dose range finding assay is conducted with eightserial dilutions covering a range of 100-0.03 μg/ml in half log steps.Definitive assays are conducted with 6-8 serial dilutions prepared inquarter log steps, centered on the expected 50% toxicity point asdetermined in the dose range finding assay. One hundred 100 μg/ml wasthe highest dose recommended to prevent false negative results from UVabsorption by the dosing solutions. One dose range finding and at leasttwo definitive trials were performed on each tetracycline derivative andcontrol compound.

Controls: Each assay included both negative (solvent) and positivecontrols. Twelve wells of negative control cultures were used on each96-well plate. Chlorpromazine (Sigma Chemicals) was used as the positivecontrol and was prepared and dosed like the test tetracyclinederivatives.

Solar Simulator: A Dermalight SOL 3 solar simulator, equipped with a UVAH1 filter (320-400 nm), was adjusted to the appropriate height.Measurement of energy through the lid of a 96-well microtiter plate wascarried out using a calibrated UV radiometer UVA sensor. Simulatorheight was adjusted to deliver 1.7±0.1 mW/cm² of UVA energy (resultingdose was 1 J/cm² per 10 minutes of exposure).

Phototoxicity Assay: Duplicate plates were prepared for each testmaterial by seeding 10⁴ 3T3 cells per well in complete medium 24 hoursbefore treatment. Prior to treatment, the medium was removed, and thecells washed once with 125 μl of prewarmed HBSS. Fifty μl of prewarmedHBSS were added to each well. Fifty μl of each test article dilutionwere added to the appropriate wells and the plates returned to theincubator for approximately one hour. Six wells were treated with eachdose of test or control article on each plate. Following the 1 hrincubation, the plates designated for the photo irradiation were exposed(with the lid on) to 1.7±0.1 mW/cm² UVA light for 50±2 minutes at roomtemperature resulting in an irradiation dose of 5 J/cm². Duplicateplates, designated for the measurement of cytotoxicity without light,were kept in the dark room temperature for 50±2 minutes. After the 50minute exposure period (with or without light) the test articledilutions were decanted from the plates and the cells washed once with125 μl of HBSS. One hundred μl of medium were added to all wells and thecells incubated as above for 24±1 hours.

After 24 hours of incubation, the medium was decanted and 100 μl of theNeutral Red containing medium were added to each well. The plates werereturned to the incubator and incubated for approximately 3 hours. After3 hours, the medium was decanted and each well rinsed once with 250 μlof HBSS. The plates were blotted to remove the HBSS and 100 μl ofNeutral Red Solvent were added to each well. After a minimum of 20minutes of incubation at room temperature (with shaking), the absorbanceat 550 nm was measured with a plate reader, using the mean of the blankouter wells as the reference. Relative survival was obtained bycomparing the amount of neutral red taken by each well treated with thetest article and positive control to the neutral red taken up by theaverage of the negative wells (12 wells) on the same plate. The amountof neutral red taken up by the negative control wells is considered tobe 100% survival.

There are several methods by which to quantify relative phototoxicity,e.g., the photoirritancy factor (PIF) and the mean photo effect (MPE),as discussed below.

Phototoxicity Determined by PIF Valuations

To determine the dose where there is a 50% decrease in relativeviability, the relative cell viability is plotted as a function ofincreasing dose and a polynomial equation is calculated to produce the“best fit” line through all the points. The dose of a test substancecorresponding to the point where this line crosses the 50% survivalpoint is calculated (termed the Inhibitory Concentration 50% or IC₅₀)and used to compare the toxicity of the test chemical in the presenceand absence of UVA/visible light.

Phototoxicity of a tetracycline derivative can be measured by itsphotoirritancy factor (PIF). The photo irritancy factor (PIF) is theratio of the IC₅₀, value in the absence of light to the IC₅₀ value inthe presence of light. That is, the PIF was determined by comparing theIC₅₀ without UVA [IC₅₀(−UVA)] with the IC₅₀ with UVA [IC₅₀ (+UVA)]:${PIF} = \frac{{IC}_{50}\left( {- {UVA}} \right)}{{IC}_{50}\left( {+ {UVA}} \right)}$

IC₅₀ values for both the UVA exposed and non-exposed groups weredetermined whenever possible. If the two values are the same, the PIF is1 and there is no phototoxic effect. If the action of the lightincreases toxicity, the IC₅₀ with light will be lower than the IC₅₀without light, and the PIF will increase.

If IC₅₀ (+UVA) can be determined but IC₅₀(−UVA) cannot, the PIF cannotbe calculated, although the compound tested may have some level ofphototoxic potential. In this case, a “>PIF” can be calculated and thehighest testable dose (−UVA) will be used for calculation of the “>PIF.”${> {PIF}} = \frac{{maximum}\quad{{dose}\left( {- {UVA}} \right)}}{{IC}_{50}\left( {+ {UVA}} \right)}$

If both, IC₅₀ (−UVA) and IC₅₀ (+UVA) cannot be calculated because thechemical does not show cytotoxicty (50% reduction in viability) up tothe highest dose tested, this would indicate a lack of phototoxicpotential.

In calculating PIF values, the data resulting from the assay procedurecan be interpreted by different methods.

For example, during the period Mar. 2, 1999 to Apr. 16, 1999, PIF valueswere obtained using the earlier phototoxicity software and itscurve-fitting algorithms, i.e. PIF1.

Since April 1999, the 3T3 phototoxicity assay has undergone extensivevalidation, and has now been incorporated into a draft guideline by theOrganization of Economic Cooperation and Development (OECD) (DraftGuideline 432). (See Spielmann et al., The International EU/COLIPA InVitro Phototoxicity Validation Study; Results of Phase II (blind trial).Part 1: The 3T3 NRU Phototoxicity Test. Toxicology In Vitro 12:305-327(1998); and Spielmann et al., A Study on UV Filter Chemicals from AnnexVII of European Union Directive 76/768/EEC, in the In Vitro 3T3Phototoxicity Test. ATLA 26:679-708 (1998).) The new guideline followsthe same assay procedure, but provides some additional guidance in theinterpretation of the resulting data, and incorporates updated software.As used herein, the PIF value interpreted by this method is termed PIF2.

According to this updated OECD draft guideline, the IC₅₀ values aredeveloped from curves fitted to the data by a multiple boot strapalgorithm. The curve fitting and calculations of the PIF are performedby software developed under contract to the German government (ZEBET,Berlin).

In particular, since there are six wells (and therefore six relativesurvival values) for each dose, the software performs multiplecalculations of the best fit line using what is called boot strapping.This approach is used to account for variations in the data. From thebootstrapped curves, the software determines a mean IC₅₀ for thetreatment. The IC₅₀ is used to compare the toxicity of the test chemicalin the presence and absence of UVA/visible light. FIG. 2 shows anexample of a set of dose response curves prepared for the positivecontrol chemical Chlorpromazine. The difference in the IC₅₀ values canbe clearly seen in this example of a highly phototoxic chemical.

Using the original software and evaluation procedures, if both IC₅₀values can be determined, the cut off value of the factor todiscriminate between phototoxicants and non-phototoxicants is a factorof 5. A factor greater than 5 is indicative of phototoxic potential ofthe test material. Using this software, the mean PIF1 for COL 10 wasdetermined to be 1.83. The mean PIF1 for COL 1002 was determined to be1.12.

The OECD draft guideline has revised the values for the PIF used todifferentiate between phototoxins, potential phototoxins andnon-phototoxins. A PIF2 of less than 2 is considered non-phototoxic, 2to less than 5 is considered potentially phototoxic, and 5 or greater isconsidered clearly phototoxic. In accordance with the OECD draftguideline, the mean PIF2 values of COL 10 and COL 1002 are 2.04 and1.35, respectively.

Phototoxicity Determined by MPE Valuations

At each data point, a photo effect is calculated according to thefollowing formula:Photo Effect_(c)=Dose Effect_(c)×Response Effect_(c) (i.e., PE _(c) =DE_(c) ×RE _(c))where c represents one concentration

Dose Effect_(c) compares the dose required to achieve percent survival nwithout UVA (c) with the dose required to achieve the same percentsurvival with UVA (c′):${{Dose}\quad{Effect}_{n}} = \frac{\begin{matrix}\left( {{{Dose}^{\prime}\left( {- {UVA}} \right)}\quad{to}\quad{give}\quad{survival}\quad{n/}} \right. \\{\left. {{Dose}\quad\left( {+ {UVA}} \right)\quad{to}\quad{give}\quad{survial}\quad n} \right) - 1}\end{matrix}}{\begin{matrix}\left( {{Dose}\quad\left( {- {UVA}} \right)\quad{to}\quad{give}\quad{survival}\quad{n/}} \right. \\{\left. {{Dose}\quad\left( {+ {UVA}} \right)\quad{to}\quad{give}\quad{survival}\quad n} \right) + 1}\end{matrix}}$As the ratio increases, the Dose Effect term approaches 1.

In the example in FIG. 3, the Dose Effect is calculated for one point.The dose of 0.4 dose units is required to reduce cell viability (termedresponse on the y axis) to 66% in the absence of light while only 0.16dose units are required to similarly reduce viability in the presence oflight. The dose effect for 0.4 dose units is:${DE}_{0.4} = {\frac{{\left( {0.4/0.16} \right) - 1}}{{\left( {0.4/0.16} \right) + 1}} = 0.43}$

The Response Effect at dose c compares the percent survival with andwithout UVA at that dose and normalizes for the total range of theresponse over the range of doses evaluated (n₁ to n_(i)).${{Response}\quad{Effect}_{c}} = \frac{{{R\left( {- {UVA}} \right)}c} - {{R\left( {+ {UVA}} \right)}c}}{R_{0}}$

where R₀ is the Total Survival Range (up to 1000%), R(−UVA)c is thesurvival without UVA at dose c, and R(+UVA)c is the survival with UVA atdose c.

As the difference between the survival without UVA at dose c and thesurvival with UVA at dose c [ie., R(−UVA)c−R(+UVA)c] increases(indicative of phototoxic potential), then the Response Effect_(c)approaches 1.0.

Again in FIG. 3, the Response Effect for the 0.4 dose is:RE _(0.4)=(66%−11%)/100%=0.55The PE in this example is PE_(0.4)=0.43*0.55=0.24

The Mean Photo Effect is the mean of the individual Photo Effect valuesover the range evaluated. It is produced from the formula:${MPE} = \frac{\sum\limits_{i = 1}^{n}{w_{i}*{PE}_{ci}}}{\sum\limits_{i = 1}^{n}w_{i}}$were w_(i) is a weighting factor for the highest viability observed foreach curve.

The MPE value is used to determine phototoxic potential. In the originalanalysis of the validation data, a material was considered nonphototoxicif the MPE was <0.1 (this includes negative MPE values) and phototoxicif the MPE was ≧0.1 (Spielmann et al, 1998). This cut off wasre-examined once the software had been rewritten and the weightingfactor added. In the draft Organization for Economic Cooperation andDevelopment phototoxicity test guideline (Guideline 432), MPE values of<0.1 (including negative values) are considered indicative of anonphototoxin, values of 0.1 to <0.15 are considered probablephototoxins, and greater than and equal to 0.15 clear phototoxins. Thisguideline is expected to become the standard after final approval in2003. The software used to calculate the MPE values is part of thisguideline.

The following table shows the phototoxicity values for severaltetracycline derivatives. The positive control is chlorpromazine. Thephototoxicity is evaluated in terms of MPE and in terms of PIF using thenew OECD draft guideline.

Example 38

The following example demonstrates a response of selected fungi toCMT-3, 4, 7, 8, and the following derivivatives of CMT-3: 302, 303, 306,308, 309 and 315.

The following fungi were inoculated onto potato dextrose agar (PDA) fromstock cultures and incubated aerobically at 30° C.: Aspergillusfumigatus ATCC 1022, Penicillium sp. (laboratory isolate), Candidaalbicans, ATCC 14053, and Rhizopus sp.

A sterile cotton tipped applicator was moistened with sterile 0.9%saline and rolled over the surface of PDA slants of Aspergillusfumigatus, Rhizopus sp. and Penicillium sp. which demonstrated copiousconidiogenesis. The conidia were suspended in 0.9% saline and theturbidity was adjusted to match a 0.5 MacFarland standard (equivalent toapproximately 1.5×10⁸ cells). Candida albicans was suspended in salineand adjusted to 0.5 MacFarland in a similar manner. These suspensionswere diluted 1:100 in sterile 0.9% saline.

SABHI Agar (Difco) pH 7.0 was prepared in 100 ml amounts and sterilizedat 121° C. for 15 min. After the SABHI agar base cooled to 50°, 10 ml ofeach of the CMT substances were prepared in 10% DMSO at a concentrationof 250 μg/ml. The CMT substances were than added at a finalconcentration of 25 μg/ml of agar base.

SABHI Agar plates of each CMT and SAHBI agar without CMT usingdimethylsulphoxide (DMSO) as a control were inoculated with 10 μl ofconidia suspension of Aspergillus fumigatus, Penicillium sp. andRhizopus sp. and 10 μl suspension of Candida albicans prepared asdescribed above. The plates were then incubated aerobically for 24 hourand for 48 hours at 30° C.

The results are set forth in Table 1 (24 hr. incubation) and Table 2 (48hr. incubation). The score table used for Tables 1 and 2 is set forth inTable 3. TABLE 1 Growth at 25 μg/ml compared to control at 24 hrsincubation Organism 3 4 7 8 302 303 306 308 309 315 DMSO AspergillusFumigatus 0 0 0 ± 0 ± 1 0 ± 0 3 Penicillium Sp. 0 3 3 3 3 3 3 0 3 0 4Rhizopus sp. 3 4 4 4 4 4 4 4 4 1 4 Candida Albicans 1 1 0 4 4 3 3 4 4 04

TABLE 2 Growth at 25 μg/ml compared to control at 48 hours Organism 3 47 8 302 303 306 308 309 315 DMSO Aspergillus Fumigatus 4 1 4 4 4 4 4 1 40 4 Penicillium Sp. 4 0 4 4 4 4 4 0 4 0 4 Rhizopus sp. 1 4 3 4 4 4 4 4 41 4 Candida Albicans 4 4 0 4 4 4 4 4 4 0 4

TABLE 3 Inhibition Score and Grading of Fungal Growth Growth InhibitionGrade Score Description 4  0% Level of growth in the absence ofanti-fungal agent (control). 3 25% 25% reduction in growth of coloniescompared to control. 2 50% 50% reduction in growth of colonies comparedto control. 1 75% 75% reduction in growth in colonies compared tocontrol. 0 100%  complete inhibition of growth.

CMT-315 yielded the best results with activity against all the fungitested. CMT-308 demonstrated activity against Aspergillus fumigatus andPenicillium sp. CMT-4 demonstrated activity against Penicillium sp., andAspergillus f. CMT-7 demonstrated strong activity against Candidaalbicans. CMT-3 inhibited Rhizopus sp., which is the most rapidlygrowing of the fungi, and can cause Rhinocerebral infection, pulnonaryinfection, mycotic keratitis, intraocular infection, orbital cellulitis,deep wound infection, external otomycosis, dermatitis, etc.

Example 39

This example demonstrates a direct comparison between CMT-3 andAmphotericin B (AmB), a conventional anti-fungal agent, in theinhibition of Aspergillus f. The plates were prepared as describedabove, using 0.125, 0.5, 0.50, 1.00 and 2.00 concentrations of each ofthe drugs tested. DMSO was used as a control

The results are shown in Table 4 below. The results were gradedaccording to the criteria set forth in Table 3. TABLE 4 Conc. (μg/ml)0.125 0.25 0.50 1.00 2.00 CMT-3 4 2 1 ±0 0 AmB 4 2 1 0 0

The results demonstrate that at various concentrations, the CMT-3inhibited growth of Aspergillus f. as effective as AmB. At aconcentration of 1.0 μg/ml, AmB inhibited 100% of fungal growth, whileCMT-3 inhibited 95% of growth. At 2.0 μg/ml, both AmB and CMT-3inhibited 100% of growth. Importantly, unlike AmB, CMT-3 demonstratesvery little toxicity in vivo at 2.0 μg/ml concentration.

Example 40

This example demonstrates the concentration of anti-fungal agentrequired to reduce the growth of the fungus by 50% in vitro (IC50) andthe minimum concentration required to completely inhibit the growth ofthe fungus in vitro (MIC). CMTs utilized in the method of the invention,i.e CMT-3 and CMT-8 were compared to Doxycycline and Amphotericin B onmicroplate agar gels.

Each drug was dissolved in DMSO (1.0 mg/ml) as a stock solution andstored at −20° C. Just prior to use, each stock solution was thawed anddiluted in DMSO to produce 6 different 100× concentrations. Potatodextrose agar was dissolved in distilled water (39 g/L) and sterilizedat 138° C. (250° F.) for 15 min. The agar solution was mixed with eachdrug (in a water bath at 60° C.) to make a series of finalconcentrations, i.e. 0.00, 0.25, 0.50, 1.00, 2.00, 4.00 μg/ml. Themixtures were then transferred to 24-well plates (1 ml/well). After thegel had formed, the fungus in PBS (spore count=1-5×10⁴/ml) wasinoculated by pipetting 10 μl onto each gel. The plates were incubatedat 30° C. for different times, depending on the requirement of eachspecies, e.g. 24 hours for Penicillium, Rhizopus, Tricothecium,Ulocladium, Absidia, Aspergilus, Caindida, Cunninghamella, 3 days forScedosporium, and 5 days for Fonsecae and Phialophora.

The MICs and IC50s for the 11 different fungi are set forth in Table 5.“*” indicates better than or similar results to Amphotericin B. “NI”indicates no detectable inhibition. TABLE 5 IC50(μg/ml) MIC(μg/ml)Candida Albicans AmB 0.5 1.0 CMT-3 1.0 2.0 CMT-8 NI NI Doxy NI NIRhizopus Species AmB 0.4 1.0 CMT-3 0.8 2.0 CMT-8 NI NI Doxy NI NIAspergilus Fumigatus AmB 0.8 2.0 CMT-3 0.5 1.0* CMT-8 NI NI Doxy NI NIPenicillium Species AmB 0.12 0.25 CMT-3 0.2 0.5 CMT-8 2.0 >4 Doxy NI NIAbsidia Species AmB 1.0 4.0 CMT-3 1.5 4.0* CMT-8 NI NI Doxy NI NIScedosporium Apiospermum AmB 4.0 >4 CMT-3 0.2 1.5* CMT-8 2.0 >4 Doxy NINI Phialophora Verrucosa AmB NI NI CMT-3 1.5 4.0* CMT-8 NI NI Doxy NI NICunninghamella Species AmB NI NI CMT-3 2.0 4.0* CMT-8 NI NI Doxy NI NITricothecium Species AmB NI NI CMT-3 0.2 1.5* CMT-8 0.7 2.0 Doxy 4.0 >4Ulocladium Species AmB 1.0 2.0 CMT-3 0.25 1.0* CMT-8 2.0 >4 Doxy NI NIFonsecae Species AmB 4.0 >4.0 CMT-3 1.0 4.0* CMT-8 NI NI Doxy NI NI

Thus, CMT-3 was effective on all 11 tested fungi, and CMT-8 had effectson some of these fungi. However, for 8 fungi out of the 11 differentspecies of fungi, Amphotericin B showed the same or less antifungalactivity than CMT-3. Doxy had essentially no detectable antifungalactivity in this experiment.

Example 41

This example demonstrates the antifungal activity of CMT-3 andAmphotericin B in vitro as being fungistatic (i.e. arresting the growthof the fungus) or fungicidal (i.e. killing the fungus).

In the pre-treatment phase of the experiment, Penicillium spores weresuspended in PBS to achieve a spore count of 10⁷/ml. CMT-3 andAmphotericin B were dissolved in DMSO to reach a concentration of 1.0mg/ml as stock solutions. 10 or 50 μl aliquots of these stock solutionswere added to the incubation mixture (containing 1.0 ml of 10⁷/ml ofPenicillium spores in PBS) to achieve a final concentration of 10 μg/mlor 50 μg/ml, respectively, for both drugs. The various incubations ofPenicillium were carried out for 24 hours at 30° C.

After the pre-treatment phase, the reaction mixtures were diluted 1000times with PBS, reducing the concentration of both drugs to 0.01 μg/mlor 0.05 μg/ml, and reducing the Penicillium spore count to 10⁴/ml. Thesedrug concentrations of both CMT-3 and Amphotericin B would not beexpected to inhibit the growth of the viable Penicillium spores.

Controls were then prepared. Before incubation, each tube was either notdiluted further, or diluted to ½ or ¼ with PBS to produce tubes withthree different spore counts, ie, 10⁴/ml, or 0.5×10⁴/ml, or 0.25×10⁴/ml.These cultures were then inoculated on potato dextrose agar gels in24-well plates, and incubated at 30° C. for 48 hours to determine therate of growth of the fungus as described before.

The controls were prepared from the suspension in the pre-treatmentphase containing only Penicillium spores 10⁷/ml, and PBS. This controlwas diluted by 1000 times with PBS to produce a spore count of10^(4/)ml. 1.0 ml of this diluted spore suspension was added to eighttubes. The stock solutions of CMT-3 and Amphotericin B, and DMSO werealso diluted by 1000 times with PBS (the new concentration being 1.0μg/ml for both drugs and 0.1% for DMSO), and 10 or 50 μl of thesesolutions was added into the above tubes. The final concentrations ineach tube was either 0.01 μg/ml or 0.05 μg/ml for both drugs (CMT-3 orAmB), or 0.001% or 0.005% for DMSO. These tubes were further treated asdescribed above to determine the growth of the fungus as controls.

The results demonstrated that all controls, including the concentrationof 0.01 and 0.05 μl/ml of both drugs (CMT-3 and Amphotericin B), showedthe same growth rate of Penicillium as the cultures without drugs,demonstrating that these low concentrations of both drugs did notinhibit the growth of the fungus in these control cultures.

Cultures of the Penicillium, after pretreatment with 10 and 50 μl/ml ofAmphotericin B, showed the same rate of growth as PBS and DMSO controlsduring the subsequent incubation phase of the experiment, indicatingthat this drug did not kill the spores during the pre-treatment phase.

In contrast, cultures of Penicillium after pretreatment with 10 and 50μl/ml of CMT-3, showed little or no growth on the agar gels comparedwith the controls, demonstrating that CMT-3 did kill the fungal sporesduring the pre-treatment phase.

Thus, Amphotericin B exhibited fungistatic activity, i.e. fungal growthwas arrested but the fugal spores were not killed. On the other hand,CMT-3 exhibited fungicidal activity against Penicillium, killing thefungus. PHOTOTOXICITY VALUES COMPOUND MPE PIF 1 PIF 2 Chloipromazine0.639 N/D 40.38 Tetracycline 0.340 5.38 N/A Doxycycline 0.522 23.3726.71 Minocycline 0.041 2.04 N/A COL 10 0.099 1.82 2.04 COL 1 0.460 N/DN/A COL 2 0.005 N/D N/A COL 3 0.654 647 84.72 COL 302 0.378 23.16 23.32COL 303 0.309 5.27 13.82 COL 305 0.420 N/D N/A COL 306 0.038 1.64 1.56COL 307 0.056 1.17 N/A COL 308 0.015 1.0 N/A COL 309 0.170 5.17 12.87COL 311 0.013 1.0 N/A COL 312 0.442 62.67 75.11 COL 313 0.462 80.2758.22 COL 314 0.475 41.1 89.48 COL 315 0.276 15.8 35.30 COL 4 0.570 N/DN/A COL 5 0.186 N/D N/A COL 6 0.155 N/D N/A COL 7 0.531 N/D N/A COL 80.703 165 82.61 COL 801 −0.001 1.0 N/A COL 802 −0.123 1.0 N/A COL 8030.047 N/D N/A COL 804 0.003 1.0 N/A COL 805 0.022 1.0 N/A COL 807 0.38240.4 N/A COL 808 0.387 46.45 N/A COL 809 0.420 N/D N/A COL 9 0.546 N/DN/A COL 1001 0.025 N/D N/A COL 1002 0.040 1.0 1.35N/A indicates that the IC₅₀ value could not be determined for the UVAexposed and/or non-exposed groupsN/D indicates that the PIF1 was not determined for the particularcompound, or was N/A as defined above.

In the present specification, some of the compounds of the invention arereferred to by codes names. The correspondence between the compound andcodes names are as follows: CHEMICAL NAMES OF THE COL COMPOUNDS COL-14-dedimethylaminotetracycline COL-36-demethyl-6-deoxy-4-dedimethylaminotetracycline COL-3017-bromo-6-demethyl-6-deoxy-4-dedimethylaminotetracycline COL-3027-nitro-6-demethyl-6-deoxy-4-dedimethylaminotetracycline COL-3039-nitro-6-demethyl-6-deoxy-4-dedimethylaminotetracycline COL-3047-acetamido-6-demethyl-6-deoxy-4-dedimethylaminotetracycline COL-3059-acetamido-6-demethyl-6-deoxy-4-dedimethylaminotetracycline COL-3069-dimethylamino-6-demethyl-6-deoxy-4-dedimethylaminotetracycline COL-3077-amino-6-demethyl-6-deoxy-4-dedimethylaminotetracycline COL-3089-amino-6-demethyl-6-deoxy-4-dedimethylaminotetracycline COL-3099-dimethylaminoacetamido-6-demethyl-6-deoxy-4-dedimethylaminotetracyclineCOL-310 7-dimethylamino-6-demethyl-6-deoxy-4-dedimethylaminotetracyclineCOL-311 9-palmitamide-6-demethyl-6-deoxy-4-dedimethylaminotetracyclineCOL-3122-CONHCH₂-pyrrolidin-1-yl-6-demethyl-6-deoxy-4-dedimethylaminotetracyclineCOL-3132-CONHCH₂-piperidin-1-yl-6-demethyl-6-deoxy-4-dedimethylaminotetracyclineCOL-3142-CONHCH₂-morpholin-1-yl-6-demethyl-6-deoxy-4-dedimethylaminotetracyclineCOL-3152-CONHCH₂-piperazin-1-yl-6-demethyl-6-deoxy-4-dedimethylaminotetracyclineCOL-4 7-chloro-4-dedimethylaminotetracycline COL-5 tetracycline pyrazoleCOL-6 4-hydroxy-4-dedimethylaminotetracycline COL-74-dedimethylamino-12α-deoxytetracycline COL-84-dedimethylaminodoxycycline COL-8019-acetamido-4-dedimethylaminodoxycycline COL-8029-dimethylaminoacetamido-4-dedimethylaminodoxycycline COL-8039-palmitamide-4-dedimethylaminodoxycycline COL-8049-nitro-4-dedimethylaminodoxycycline COL-8059-amino-4-dedimethylaminodoxycycline COL-8069-dimethylamino-4-dedimethylaminodoxycycline COL-8072-CONHCH₂-pyrrolidin-1-yl-4-dedimthylaminodoxycycline COL-8082-CONHCH₂-piperidin-1-yl-4-dedimethylaminodoxycycline COL-8092-CONHCH₂-piperazin-1-yl-4-dedimethylaminodoxycycline COL-104-dedimethylaminominocycline (a.k.a. COL-310) COL-10017-trimethylammonium-4-dedimethylaminosancycline COL-10029-nitro-4-dedimethylaminominocycline

Index Of Structure

wherein R7 is selected from the group consisting of hydrogen, amino,nitro, mono(lower alkyl)amino, halogen, di(lower alkyl)amino,ethoxythiocarbonylthio, azido, acylamino, diazonium, cyano, andhydroxyl; R6-a is selected from the group consisting of hydrogen andmethyl; R6 and R5 are selected from the group consisting of hydrogen andhydroxyl; R8 is selected from the group consisting of hydrogen andhalogen; R9 is selected from the group consisting of hydrogen, amino,azido, nitro, acylamino, hydroxy, ethoxythiocarbonylthio, mono(loweralkyl)amino, halogen, diazonium, di(lower alkyl)amino and RCH(NH₂)CO; Ris hydrogen or lower alkyl; and pharmaceutically acceptable andunacceptable salts thereof; with the following provisos: when either R7and R9 are hydrogen then R8 must be halogen; and when R6-a, R6, R5 andR9 are all hydrogen and R7 is hydrogen, amino, nitro, halogen,dimethylamino or diethylamino, then R8 must be halogen; and when R6-a ismethyl, R6 and R9 are both hydrogen, R5 is hydroxyl and R7 is hydrogen,amino, nitro, halogen or diethylamino, then R8 is halogen; and when R6-ais methyl, R6 is hydroxyl, R5, R7 and R9 are all hydrogen, then R8 mustbe halogen; and when R6-a, R6 and R5 are all hydrogen, R9 is methylaminoand R7 is dimethylamino, then R8 must be halogen; and when R6-a ismethyl, R6 is hydrogen, R5 is hydroxyl, R9 is methylamino and R7 isdimethylamino, then R8 must be halogen; and when R6-a is methyl, R6, R5and R9 are all hydrogen and R7 is cyano, then R8 must be halogen.

wherein R7 is selected from the group consisting of hydrogen, amino,nitro, mono(lower alkyl)amino, halogen, di(lower alkyl)amino,ethoxythiocarbonylthio, azido, acylamino, diazonium, cyano, andhydroxyl; R6-a is selected from the group consisting of hydrogen andmethyl; R6 and R5 are selected from the group consisting of hydrogen andhydroxyl; R4 is selected from the group consisting of NOH, N—NH-A, andNH-A, where A is a lower alkyl group; R8 is selected from the groupconsisting of hydrogen and halogen; R9 is selected from the groupconsisting of hydrogen, amino, azido, nitro, acylamino, hydroxy,ethoxythiocarbonylthio, mono(lower alkyl)amino, halogen, di(loweralkyl)amino and RCH(NH₂)CO; R is hydrogen or lower alkyl; andpharmaceutically acceptable and unacceptable salts thereof; with thefollowing provisos: when R4 is NOH, N—NH-alkyl or NH-alkyl and R7, R6-a,R6, R5, and R9 are all hydrogen, then R8 must be halogen; and when R4 isNOH, R6-a is methyl, R6 is hydrogen or hydroxyl, R7 is halogen, R5 andR9 are both hydrogen, then R8 must be halogen; and when R4 isN—NH-alkyl, R6-a is methyl, R6 is hydroxyl and R7, R5, R9 are allhydrogen, then R8 must be halogen; and when R4 is NH-alkyl, R6-a, R6, R5and R9 are all hydrogen, R7 is hydrogen, amino, mono(lower alkyl)amino,halogen, di(lower alkyl)amino or hydroxyl, then R8 must be halogen; andwhen R4 is NH-alkyl, R6-a is methyl, R6 and R9 are both hydrogen, R5 ishydroxyl, and R7 is mono(lower alkyl)amino or di(lower alkyl)amino, thenR8 must be halogen; and when R4 is NH-alkyl, R6-a is methyl, R6 ishydroxy or hydrogen and R7, R5, and R9 are all be hydrogen, then R8 mustbe halogen.

General Formula (I)

wherein R7, R8, and R9 taken together in each case, have the followingmeanings: R7 R8 R9 azido hydrogen hydrogen dimethylamino hydrogen azidohydrogen hydrogen amino hydrogen hydrogen azido hydrogen hydrogen nitrodimethylamino hydrogen amino acylamino hydrogen hydrogen hydrogenhydrogen acylamino amino hydrogen nitro hydrogen hydrogen(N,N-dimethyl)glycylamino amino hydrogen amino hydrogen hydrogenethoxythiocarbonylthio dimethylamino hydrogen acylamino dimethylaminohydrogen diazonium dimethylamino chloro amino hydrogen chloro aminoamino chloro amino acylamino chloro acylamino amino chloro hydrogenacylamino chloro hydrogen inonoalkylamino chloro amino nitro chloroamino dimethylamino chloro acylamino dimethylamino chloro dimethylaminodimethylamino hydrogen hydrogen hydrogen hydrogen dimethylamino andGeneral Formula (II)

Structure L

Structure M

Structure N

Structure O

wherein R7, R8, and R9 taken together in each case, have the followingmeanings: R7 R8 R9 azido hydrogen hydrogen dimethylamino hydrogen azidohydrogen hydrogen amino hydrogen hydrogen azido hydrogen hydrogen introdimethylamino hydrogen amino acylamino hydrogen hydrogen hydrogenhydrogen acylamino amino hydrogen nitro hydrogen hydrogen(N,N-dimethyl)glycylamino amino hydrogen amino hydrogen hydrogenethoxytbiocarbonyithic dimethylamino hydrogen acylamino hydrogenhydrogen diazonium hydrogen hydrogen dirnethylamino diazonium hydrogenhydrogen ethoxythiocar- hydrogen hydrogen bonylthio dimethylamino chloroamino amino chloro amino acylamino chloro acylamino hydrogen chloro ammoamino chloro hydrogen acylamino chloro hydrogen monoalkyl amino chloroamino nitro chloro amino and General Formula (III)

Structure Pwherein R8 is hydrogen or halogen and R9 is selected from the groupconsisting of nitro, (N,N-dimethyl)glycylamino, andethoxythiocarbonylthio; and

General Formula (IV)

wherein R7, R8, and R9 taken together in each case, have the followingmeanings: R7 R8 R9 amino hydrogen hydrogen nitro hydrogen hydrogen azidohydrogen hydrogen dimethylamino hydrogen azido hydrogen hydrogen aminohydrogen hydrogen azido hydrogen hydrogen nitro bromo hydrogen hydrogendimethylamino hydrogen amino acylamino hydrogen hydrogen hydrogenhydrogen acylamino amino hydrogen nitro hydrogen hydrogen(N,N-dimethyl)glycylamino amino hydrogen amino diethylamino hydrogenhydrogen hydrogen hydrogen ethoxythiocarbonylthio dimethylamino hydrogenmethylamino dimethylamino hydrogen acylamino dimethylamino chloro aminoamino chloro amino acylamino chloro acylamino hydrogen chloro aminoamino chloro hydrogen acylamino chloro hydrogen monoalkylamino chloroamino nitro chloro amino hydrogen hydrogen amino hydrogen hydrogenpalmitamide and STRUCTURE L STRUCTURE M STRUCTURE N STRUCTURE Oand pharmaceutically acceptable and unacceptable salts thereof.

wherein R7 is selected from the group consisting of hydrogen, amino,nitro, mono(lower alkyl)amino, halogen, di(lower alkyl)amino,ethoxythiocarbonylthio, azido, acylamino, diazonium, cyano, andhydroxyl; R6-a is selected from the group consisting of hydrogen andmethyl; R6 and R5 are selected from the group consisting of hydrogen andhydroxyl; R8 is selected from the group consisting of hydrogen andhalogen; R9 is selected from the group consisting of hydrogen, amino,azido, nitro, acylamino, hydroxy, ethoxythiocarbonylthio, mono(loweralkyl)amino, halogen, diazonium, di(lower alkyl)amino and RCH(NH₂)CO; Ris hydrogen or lower alkyl; R^(a) and R^(b) are selected from the groupconsisting of hydrogen, methyl, ethyl, n-propyl and 1-methylethyl withthe proviso that R^(a) and R^(b) cannot both be hydrogen; R^(c) andR^(d) are, independently (CH₂)_(n)CHR^(e) wherein n is 0 or 1 and R^(e)is selected from the group consisting of hydrogen, alkyl, hydroxy,lower(C₁-C₃) alkoxy, amino, or nitro; and, W is selected from the groupconsisting of (CHR^(e))_(m) wherein m is 0-3 and R^(e) is as above, NH,N(C₁-C₃) straight chained or branched alkyl, O, S and N(C₁-C₄) straightchain or branched alkoxy; and pharmaceutically acceptable andunacceptable salts thereof. In a further embodiment, the followingprovisos apply: when either R7 and R9 are hydrogen then R8 must behalogen; and when R6-a, R6, R5 and R9 are all hydrogen and R7 ishydrogen, amino, nitro, halogen, dimethylamino or diethylamino, then R8must be halogen; and when R6-a is methyl, R6 and R9 are both hydrogen,R5 is hydroxyl, and R7 is hydrogen, amino, nitro, halogen ordiethylamino, then R8 is halogen; and when R6-a is methyl, R6 ishydroxyl, R5, R7 and R9 are all hydrogen, then R8 must be halogen; andwhen R6-a, R6 and R5 are all hydrogen, R9 is methylamino and R7 isdimethylamino, then R8 must be halogen; and when R6-a is methyl, R6 ishydrogen, R5 is hydroxyl, R9 is methylamino and R7 is dimethylamino,then R8 must be halogen; and when R6-a is methyl, R6, R5 and R9 are allhydrogen and R7 is cyano, then R8 must be halogen.

Structure K

wherein: R7, R8, and R9 taken together in each case, have the followingmeanings: R7 R8 R9 hydrogen hydrogen amino hydrogen hydrogen palmitamideand STRUCTURE L STRUCTURE M STRUCTURE N STRUCTURE O

wherein: R7, R8, and R9 taken together in each case, have the followingmeanings: R7 R8 R9 hydrogen hydrogen acetamido hydrogen hydrogendimethylaminoacetamido hydrogen hydrogen nitro hydrogen hydrogen aminoand STRUCTURE Pwherein: R8, and R9 taken together are, respectively, hydrogen andnitro.

Structure K

wherein: R7, R8, and R9 taken together are, respectively, hydrogen,hydrogen and dimethylamino. STRUCTURE STRUCTURE STRUCTURE STRUCTURE C DE Fwherein R7 is selected from the group consisting of an aryl, alkenyl andalkynyl; R6-a is selected from the group consisting of hydrogen andmethyl; R6 and R5 are selected from the group consisting of hydrogen andhydroxyl; R8 is selected from the group consisting of hydrogen andhalogen; R9 is selected from the group consisting of hydrogen, amino,azido, nitro, acylamino, hydroxy, ethoxythiocarbonylthio, mono(loweralkyl)amino, halogen, diazonium, di(lower alkyl)amino and RCH(NH₂)CO;and pharmaceutically acceptable and unacceptable salts thereof;

or STRUCTURE STRUCTURE STRUCTURE STRUCTURE C D E Fwherein: R7 is selected from the group consisting of hydrogen, amino,nitro, mono(lower alkyl)amino, halogen, di(lower alkyl)amino,ethoxythiocarbonylthio, azido, acylamino, diazonium, cyano, andhydroxyl; R6-a is selected from the group consisting of hydrogen andmethyl; R6 and R5 are selected from the group consisting of hydrogen andhydroxyl; R8 is selected from the group consisting of hydrogen andhalogen; R9 is selected from the group consisting of an aryl, alkenyland alkynyl; and pharmaceutically acceptable and unacceptable saltsthereof;

or STRUCTURE STRUCTURE STRUCTURE STRUCTURE C D E F

wherein: R7 and R9 are selected from the group consisting of an aryl,alkene, alkyne, or mixures thereof; R6-a is selected from the groupconsisting of hydrogen and methyl; R6 and R5 are selected from the groupconsisting of hydrogen and hydroxyl; R8 is selected from the groupconsisting of hydrogen and halogen; and pharmaceutically acceptable andunacceptable salts thereof. STRUCTURE STRUCTURE STRUCTURE STRUCTURE G HI Jwherein R7 is selected from the group consisting of an aryl, alkenyl andalkynyl; R6-a is selected from the group consisting of hydrogen andmethyl; R6 and R5 are selected from the group consisting of hydrogen andhydroxyl; R4 is selected from the group consisting of NOH, N—NH-A, andNH-A,where A is a lower alkyl group; R8 is selected from the groupconsisting of hydrogen and halogen;R9 is selected from the groupconsisting of hydrogen, amino, azido, nitro, acylamino, hydroxy,ethoxythiocarbonylthio, mono(lower alkyl)amino, halogen, di(loweralkyl)amino and RCH(NH₂)CO; and pharmaceutically acceptable andunacceptable salts thereof;

or STRUCTURE STRUCTURE STRUCTURE STRUCTURE G H I Jwherein R7 is selected from the group consisting of hydrogen, amino,nitro, mono(lower alkyl)amino, halogen, di(lower alkyl)amino,ethoxythiocarbonylthio, azido, acylamino, diazonium, cyano, andhydroxyl; R6-a is selected from the group consisting of hydrogen andmethyl; R6 and R5 are selected from the group consisting of hydrogen andhydroxyl; R4 is selected from the group consisting of NOH, N—NH-A, andNH-A, where A is a lower alkyl group; R8 is selected from the groupconsisting of hydrogen and halogen; R9 is selected from the groupconsisting of an aryl, alkenyl and alkynyl; and pharmaceuticallyacceptable and unacceptable salts thereof;

or STRUCTURE STRUCTURE STRUCTURE STRUCTURE G H I Jwherein: R7 and R9 are selected from the group consisting of an aryl,alkenyl, alkynyl; or mixtures thereof; R6-a is selected from the groupconsisting of hydrogen and methyl; R6 and R5 are selected from the groupconsisting of hydrogen and hydroxyl; R4 is selected from the groupconsisting of NOH, N—NH-A, and NH-A, where A is a lower alkyl group; andR8 is selected from the group consisting of hydrogen and halogen; andpharmaceutically acceptable and unacceptable salts thereof.

Structure K

wherein R7 is selected from the group consisting of an aryl, alkenyl andalkynyl; R8 is selected from the group consisting of hydrogen andhalogen; R9 is selected from the group consisting of hydrogen, amino,azido, nitro, acylamino, hydroxy, ethoxythiocarbonylthio, mono(loweralkyl)amino, halogen, di(lower alkyl)amino and RCH(NH₂)CO; andpharmaceutically acceptable and unacceptable salts thereof;

or

Struture K

wherein: R7 is selected from the group consisting of hydrogen, amino,nitro, mono(lower alkyl)amino, halogen, di(lower alkyl)amino,ethoxythiocarbonylthio, azido, acylamino, diazonium, cyano, andhydroxyl; R8 is selected from the group consisting of hydrogen andhalogen; R9 is selected from the group consisting of an aryl, alkenyland alkynyl; and pharmaceutically acceptable and unacceptable saltsthereof;

or

Structure K

wherein: R7 and R9 are selected from the group consisting of an aryl,alkenyl, alkynyl and mixtures thereof; and R8 is selected from the groupconsisting of hydrogen and halogen; and pharmaceutically acceptable andunacceptable salts thereof;

and STRUCTURE STRUCTURE STRUCTURE STRUCTURE L M N Owherein: R7 is selected from the group consisting of an aryl, alkenyland alkynyl; R8 is selected from the group consisting of hydrogen andhalogen; and pharmaceutically acceptable and unacceptable salts thereof;

or STRUCTURE STRUCTURE STRUCTURE STRUCTURE L M N Owherein R7 is selected from the group consisting of hydrogen, amino,nitro, mono(lower alkyl)amino, halogen, di(lower alkyl)amino,ethoxythiocarbonylthio, azido, acylamino, diazonium, cyano, andhydroxyl; R8 is selected from the group consisting of hydrogen andhalogen; R9 is selected from the group consisting of an aryl, alkenyland alkynyl; and pharmaceutically acceptable and unacceptable saltsthereof;

or STRUCTURE STRUCTURE STRUCTURE STRUCTURE L M N Owherein R7 is and R9 are selected from the group consisting of an aryl,alkenyl, alkynyl and mixtures thereof; R8 is selected from the groupconsisting of hydrogen and halogen; R9 is selected from the groupconsisting of hydrogen, amino, azido, nitro, acylamino, hydroxy,ethoxythiocarbonylthio, mono(lower alkyl)amino, halogen, di(loweralkyl)amino and RCH(NH₂)CO; and pharmaceutically acceptable andunacceptable salts thereof;and

Structure P

wherein R9 is selected from the group consisting of an aryl, alkenyl andalkynyl; and R8 is selected from the group consisting of hydrogen andhalogen; and pharmaceutically acceptable and unacceptable salts thereof;

and STRUCTURE Q STRUCTURE Rwherein R7 is selected from the group consisting of an aryl, alkenyl andalkynyl; R8 is selected from the group consisting of hydrogen andhalogen; R9 is selected from the group consisting of hydrogen, amino,azido, nitro, acylamino, hydroxy, ethoxythiocarbonylthio, mono(loweralkyl)amino, halogen, di(lower alkyl)amino and RCH(NH₂)CO; andpharmaceutically acceptable and unacceptable salts thereof;

or STRUCTURE Q STRUCTURE Rwherein R7 is selected from the group consisting of hydrogen, amino,nitro, mono(lower alkyl)amino, halogen, di(lower alkyl)amino,ethoxythiocarbonylthio, azido, acylamino, diazonium, cyano, andhydroxyl; R8 is selected from the group consisting of hydrogen andhalogen; R9 is selected from the group consisting of an aryl, alkenyland alkynyl; and pharmaceutically acceptable and unacceptable saltsthereof;

or STRUCTURE Q STRUCTURE Rwherein R7 and R9 are selected from the group consisting of an aryl,alkenyl, alkynyl; and mixtures thereof; R8 is selected from the groupconsisting of hydrogen and halogen; and pharmaceutically acceptable andunacceptable salts thereof.

Structure S-Z

wherein R7 is selected from the group consisting of an aryl, alkenyl andalkynyl; R6-a is selected from the group consisting of hydrogen andmethyl; R6 and R5 are selected from the group consisting of hydrogen andhydroxyl; R8 is selected from the group consisting of hydrogen andhalogen; R9 is selected from the group consisting of hydrogen, amino,azido, nitro, acylamino, hydroxy, ethoxythiocarbonylthio, mono(loweralkyl)amino, halogen, diazonium, di(lower alkyl)amino and RCH(NH₂)CO;R^(a) and R^(b) are selected from the group consisting of hydrogen,methyl, ethyl, n-propyl and 1-methylethyl with the proviso that R^(a)and R^(b) cannot both be hydrogen; R^(c) and R^(d) are, independently,(CH₂)_(n)CHR^(e) wherein n is 0 or 1 and R^(e) is selected from thegroup consisting of hydrogen, alkyl, hydroxy, lower(C₁-C₃)alkoxy, amino,or nitro; and, W is selected from the group consisting of (CHR^(e))_(m)wherein m is 0-3 and said R^(e) is as above, NH, N(C₁-C₃) straightchained or branched alkyl, O, S and N(C₁-C₄) straight chain or branchedalkoxy; and pharmaceutically acceptable and unacceptable salts thereof;

or

Structure S-Z

wherein R7 is selected from the group consisting of hydrogen, amino,nitro, mono(lower alkyl)amino, halogen, di(lower alkyl)amino,ethoxythiocarbonylthio, azido, acylamino, diazonium, cyano, andhydroxyl; R6-a is selected from the group consisting of hydrogen andmethyl; R6 and R5 are selected from the group consisting of hydrogen andhydroxyl; R8 is selected from the group consisting of hydrogen andhalogen; R9 is selected from the group consisting of an aryl, alkenyland alkynyl; R^(a) and R^(b) are selected from the group consisting ofhydrogen, methyl, ethyl, n-propyl and 1-methylethyl with the provisothat R^(a) and R^(b) cannot both be hydrogen; R^(a) and R^(d) are,independently, (CH₂)_(n)CHR^(e) wherein n is 0 or 1 and R^(e) isselected from the group consisting of hydrogen, alkyl, hydroxy,lower(C₁-C₃) alkoxy, amino, or nitro; and, W is selected from the groupconsisting of (CHR^(e))_(m) wherein m is 0-3 and said R^(e) is as above,NH, N(C₁-C₃) straight chained or branched alkyl, 0, S and N(C₁-C₄)straight chain or branched alkoxy; and pharmaceutically acceptable andunacceptable salts thereof;

or

Structure S-Z

wherein: R7 and R9 are selected from the group consisting of an aryl,alkenyl, alkynyl and mixtures thereof; R6-a is selected from the groupconsisting of hydrogen and methyl; R6 and R5 are selected from the groupconsisting of hydrogen and hydroxyl; R8 is selected from the groupconsisting of hydrogen and halogen; R^(a) and R^(b) are selected fromthe group consisting of hydrogen, methyl, ethyl, n-propyl and1-methylethyl with the proviso that R^(a) and R^(b) cannot both behydrogen; R^(c) and R^(d) are, independently, (CH₂)_(n)CHR^(e) wherein nis 0 or 1 and R^(e) is selected from the group consisting of hydrogen,alkyl, hydroxy, lower(C₁-C₃) alkoxy, amino, or nitro; and W is selectedfrom the group consisting of (CHR^(e))_(m) wherein m is 0-3 and saidR^(e) is as above, NH, N(C₁-C₃) straight chained or branched alkyl, O, Sand N(C₁-C₄) straight chain or branched alkoxy; and pharmaceuticallyacceptable and unacceptable salts thereof.

Throughout this specification, the descriptions of some structuresinclude the term “lower alkyl.” The term “lower alkyl” means an alkylgroup comprising relatively few carbon atoms, for example, about one toten carbon atoms. A preferred low end of this range is one, two, three,four or five carbon atoms; and a preferred high end of this range issix, seven, eight, nine or ten carbon atoms. Some examples of “loweralkyl” groups include methyl groups, ethyl groups, propyl groups,isopropyl groups, butyl groups, etc.

1. A method for simultaneously treating mucositis and fungal infectionin a mammal in need thereof, said method comprising administering tosaid mammal an effective amount of an anti-mucositis and anti-fungalpharmaceutical composition consisting of a tetracycline compound in anamount that is effective to simultaneously treat mucositis and fungalinfection, but has substantially no antibiotic activity.
 2. A methodaccording to claim 1 wherein said fungus is selected from the groupconsisting of Cryptococcus species, Candida albicans, Rhizopus species,Aspergillus fumigatus, Penicillium species, Absidia species,Scedosporium apiospermum, Phialophora verrucosa, Cunninghamella species,Tricothecium species, Ulocladium species, Fonsecae species, andcombinations thereof.
 3. A method according to claim 1 wherein saidfungus is selected from the group consisting of Rhizopus species,Absidia species, Scedosporium apiospermum, Phialophora verrucosa,Cunninghamella species, Tricothecium species, Ulocladium species,Fonsecae species, or a combination thereof, and wherein saidnon-antibiotic tetracycline derivative is CMT-3.
 4. A method accordingto claim 1 wherein said fungus is Aspergillus fumigatus, Penicilliumspecies, Rhizopus species, Candida albicans, or a combination thereof,and wherein said non-antibiotic tetracycline derivative is CMT-315.
 5. Amethod according to claim 1 wherein said fungus is Penicillium speciesand said non-antibiotic tetracycline derivative is CMT4.
 6. A methodaccording to claim 1 wherein said fungus is Candida albicans and saidnon-antibiotic tetracycline derivative is CMT-7.
 7. A method accordingto claim 1 wherein said fungus is Aspergillus fumigatus, Penicilliumspecies or a combination thereof, and said non-antibiotic is CMT-308. 8.A method according to claim 1 wherein said fungus is Penicilliulnspecies, Scedosporium apiospermum, Tricothecium species, Ulocladiumspecies, or a combination thereof and said non-antibiotic tetracyclinederivative is CMT-8.
 9. A method according to claim 1 wherein saidmammal is a human.
 10. A method according to claim 1 wherein saidtreatment comprises administering said non-antibiotic tetracyclinederivative systemically.
 11. A method according to claim 10, whereinsaid systemic administration is oral administration, intravenousinjection, intramuscular injection, subcutaneous administration,transdermal administration or intranasal administration.
 12. A methodaccording to claim 1, wherein said tetracycline compound is anantibiotic tetracycline compound administered in an amount which is10-80% of the antibiotic amount.
 13. A method according to claim 1,wherein said tetracycline compound is doxycycline administered twice aday in a dose of 20 mg.
 14. A method according to claim 1, wherein saidtetracycline compound is minocycline administered once a day in a doseof 38 mg.
 15. A method according to claim 1, wherein said tetracyclinecompound is minocycline administered twice a day in a dose of 38 mg. 16.A method according to claim 1, wherein said tetracycline compound isminocycline administered three times a day in a dose of 38 mg.
 17. Amethod according to claim 1, wherein said tetracycline compound istetracycline administered twice a day in a dose of 60 mg/day.
 18. Amethod according to claim 1, wherein said tetracycline compound istetracycline administered three times a day in a dose of 60 mg/day. 19.A method according to claim 1, wherein said tetracycline compound istetracycline administered four times a day in a dose of 60 mg/day.
 20. Amethod according to claim 1, wherein said tetracycline compound is anantibiotic tetracycline compound administered in an amount which resultsin a serum concentration which is 10-80% of the minimum antibiotic serumconcentration.
 21. A method according to claim 1, wherein saidtetracycline compound is doxycycline administered in an amount whichresults in a serum concentration which is 1.0 μg/ml.
 22. A methodaccording to claim 1, wherein said tetracycline compound is minocyclineadministered in an amount which results in a serum concentration whichis 0.8 μg/ml.
 23. A method according to claim 1, wherein saidtetracycline compound is tetracycline administered in an amount whichresults in a serum concentration which is 0.5 μg/ml.
 24. A methodaccording to claim 12 or 20, wherein said antibiotic tetracyclinecompound is doxycycline, minocycline, tetracycline, oxytetracycline,chlortetracycline, demeclocycline or pharmaceutically acceptable saltsthereof.
 25. A method according to claim 24, wherein said antibiotictetracycline compound is doxycycline.
 26. A method according to claim25, wherein said doxycycline is administered in an amount which providesa serum concentration in the range of about 0.1 to about 0.8 μg/ml. 27.A method according to claim 25, wherein said doxycycline is administeredin an amount of 20 milligrams twice daily.
 28. A method according toclaim 26, wherein said doxycycline is administered by sustained releaseover a 24 hour period.
 29. A method according to claim 28, where saiddoxcycline is administered in an amount of 40 milligrams.
 30. A methodaccording to claim 1, wherein said tetracycline compound is anon-antibiotic tetracycline compound.
 31. A method according to claim30, wherein said non-antibiotic tetracycline compound is:4-de(dimethylamino)tetracycline (CMT-1), tetracyclinonitrile (CMT-2),6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline (CMT-3),4-de(dimethylamino)-7-chlorotetracycline (CMT-4), tetracycline pyrazole(CMT-5) 4-hydroxy-4-de(dimethylamino)tetracycline (CMT-6),4-de(dimethylamino)-12α-deoxytetracycline (CMT-7),6-α-deoxy-5-hydroxy-4-de(dimethylamino)tetracycline (CMT-8),4-de(dimethylamino)-12α-deoxyanhydrotetracycline (CMT-9), or4-de(dimethylamino)minocycline (CMT-10).
 32. A method according to claim30, wherein the non-antibiotic tetracycline compound is selected fromthe group consisting of:

wherein: R7 is selected from the group consisting of hydrogen, amino,nitro, mono(lower alkyl)amino, halogen, di(lower alkyl)amino,ethoxythiocarbonylthio, azido, acylamino, diazonium, cyano, andhydroxyl; R6-a is selected from the group consisting of hydrogen andmethyl; R6 and R5 are selected from the group consisting of hydrogen andhydroxyl; R8 is selected from the group consisting of hydrogen andhalogen; R9 is selected from the group consisting of hydrogen, amino,azido, nitro, acylamino, hydroxy, ethoxythiocarbonylthio, mono(loweralkyl)amino, halogen, diazonium, di(lower alkyl)amino and RCH(NH₂)CO; Ris hydrogen or lower alkyl; and pharmaceutically acceptable saltsthereof; with the following provisos: when either R7 and R9 are hydrogenthen R8 must be halogen; and when R6-a, R6, R5 and R9 are all hydrogenand R7 is hydrogen, amino, nitro, halogen, dimethylamino ordiethylamino, then R8 must be halogen; and when R6-a is methyl, R6 andR9 are both hydrogen, R5 is hydroxyl, and R7 is hydrogen, amino, nitro,halogen or diethylamino, then R8 is halogen; and when R6-a is methyl, R6is hydroxyl, R5, R7 and R9 are all hydrogen, then R8 must be halogen;and when R6-a, R6 and R5 are all hydrogen, R9 is methylamino and R7 isdimethylamino, then R8 must be halogen; and when R6-a is methyl, R6 ishydrogen, R5 is hydroxyl, R9 is methylamino and R7 is dimethylamino,then R8 must be halogen; and when R6-a is methyl, R6, R5 and R9 are allhydrogen and R7 is cyano, then R8 must be halogen.
 33. A methodaccording to claim 30, wherein the non-antibiotic tetracycline compoundis selected from the group consisting of:

wherein: R7 is selected from the group consisting of hydrogen, amino,nitro, mono(lower alkyl)amino, halogen, and di(lower alkyl)amino,ethoxythiocarbonylthio, azido, acylamino, diazonium, cyano, andhydroxyl; R6-a is selected from the group consisting of hydrogen andmethyl; R6 and R5 are selected from the group consisting of hydrogen andhydroxyl; R4 is selected from the group consisting of NOH, N—NH-A, andNH-A, where A is a lower alkyl group; R8 is selected from the groupconsisting of hydrogen and halogen; R9 is selected from the groupconsisting of hydrogen, amino, azido, nitro, acylamino, hydroxy,ethoxythiocarbonylthio, mono(lower alkyl)amino, halogen, di(loweralkyl)amino and RCH(NH₂)CO; R is hydrogen or lower alkyl; andpharmaceutically acceptable salts thereof; with the following provisos:when R4 is NOH, N—NH-alkyl or NH-alkyl and R7, R6-a, R6, R5, and R9 areall hydrogen, then R8 must be halogen; and when R4 is NOH, R6-a ismethyl, R6 is hydrogen or hydroxyl, R7 is halogen, R5 and R9 are bothhydrogen, then R8 must be halogen; and when R4 is N—NH-alkyl, R6-a ismethyl, R6 is hydroxyl and R7, R5, R9 are all hydrogen, then R8 must behalogen; and when R4 is NH-alkyl, R6-a, R6, R5 and R9 are all hydrogen,R7 is hydrogen, amino, mono(lower alkyl)amino, halogen, di(loweralkyl)amino or hydroxyl, then R8 must be halogen; and when R4 isNH-alkyl, R6-a is methyl, R6 and R9 are both hydrogen, R5 is hydroxyl,and R7 is mono(lower alkyl)amino or di(lower alkyl)amino, then R8 mustbe halogen; and when R4 is NH-alkyl, R6-a is methyl, R6 is hydroxy orhydrogen and R7, R5, and R9 are all be hydrogen, then R8 must behalogen.
 34. A method according to claim 30 wherein the non-antibiotictetracycline compound is selected from the group consisting of:

wherein: R7, R8, and R9 taken together in each case, have the followingmeanings: R7 R8 R9 azido hydrogen hydrogen dimethylamino hydrogen azidohydrogen hydrogen amino hydrogen hydrogen azido hydrogen hydrogen nitrodimethylamino hydrogen amino acylamino hydrogen hydrogen hydrogenhydrogen acylainino amino hydrogen nitro hydrogen hydrogen(N,N-dimethyl)glycylamino amino hydrogen amino hydrogen hydrogenethoxytliiocarbonylthio dimethylamino hydrogen acylamino dimethylaminohydrogen diazonium diniethylamino chloro amino hydrogen chloro aminoamino chloro amino acylainmo chloro acylamino amino chloro hydrogenacylamino chloro hydrogen monoalkylamino chloro amino nitro chloro aminodimethylamino chloro acylamino dimethylamino chloro dimethylaminohydrogen hydrogen dimethylamino dimethylamino hydrogen hydrogen and

Structure L

Structure M

Structure N

Structure O

wherein: R7, R8, and R9 taken together in each case, have the followingmeanings: R7 R8 R9 azido hydrogen hydrogen dimethylamino hydrogen azidohydrogen hydrogen amino hydrogen hydrogen azido hydrogen hydrogen nitrodimethylamino hydrogen amino acylamino hydrogen hydrogen hydrogenhydrogen acylamino amino hydrogen intro hydrogen hydrogen(N,N-dimethyl)glycylamino amino hydrogen amino hydrogen hydrogenethoxythiocarbonylthio dimethylamino hydrogen acylainino hydrogenhydrogen diazonium hydrogen hydrogen dimethylamino diazonium hydrogenhydrogen ethoxythiocar- hydrogen hydrogen bonylthio dimethylamino chloroamino amino chloro ammo acylamino chloro acylamino hydrogen chloro ammoamino chloro hydrogen acylamino chloro hydrogen monoalkylammo chloroamino nitro chloro amino and

Structure P

wherein: R8 is hydrogen or halogen and R9 is selected from the groupconsisting of nitro, (N,N-dimethyl)glycylamino, andethoxythiocarbonylthio; and

wherein: R7, R8, and R9 taken together in each case, have the followingmeanings: R7 R8 R9 amino hydrogen hydrogen nitro hydrogen hydrogen azidohydrogen hydrogen dimethylamino hydrogen azido hydrogen hydrogen aminohydrogen hydrogen azido hydrogen hydrogen nitro bromo hydrogen hydrogendimethylamino hydrogen amino acylamino hydrogen hydrogen hydrogenhydrogen acylamino amino hydrogen nitro hydrogen hydrogen(N,N-dimethyl)glycylamino amino hydrogen amino diethylamino hydrogenhydrogen hydrogen hydrogen ethoxythiocarbonylthio dimethylamino hydrogenmethylamino dimethylamino hydrogen acylamino dimethylamino chloro aminoamino chloro amino acylamino chloro acylamino hydrogen chloro aminoamino chloro hydrogen acylamino chloro hydrogen monoalkylamino chloroamino nitro chloro amino

and pharmaceutically acceptable salts thereof
 35. A method according toclaim 30, wherein the non-antibiotic tetracycline compound is selectedfrom the group consisting of:

wherein: R7 is selected from the group consisting of hydrogen, amino,nitro, mono(lower alkyl)amino, halogen, di(lower alkyl)amino,ethoxythiocarbonylthio, azido, acylamino, diazonium, cyano, andhydroxyl; R6-a is selected from the group consisting of hydrogen andmethyl; R6 and R5 are selected from the group consisting of hydrogen andhydroxyl; R8 is selected from the group consisting of hydrogen andhalogen; R9 is selected from the group consisting of hydrogen, amino,azido, nitro, acylamino, hydroxy, ethoxythiocarbonylthio, mono(loweralkyl)amino, halogen, diazonium, di(lower alkyl)amino and RCH(NH₂)CO; Ris hydrogen or lower alkyl; R^(a) and R^(b) are selected from the groupconsisting of hydrogen, methyl, ethyl, n-propyl and 1-methylethyl withthe proviso that R^(a) and R^(b) cannot both be hydrogen; R^(c) andR^(d) are, independently, (CH₂)_(n)CHR^(e) wherein n is 0 or 1 and R^(e)is selected from the group consisting of hydrogen, alkyl, hydroxy,lower(C₁-C₃) alkoxy, amino, or nitro; and, W is selected from the groupconsisting of (CHR^(e))_(m) wherein m is 0-3 and said R^(e) is selectedfrom the group consisting of hydrogen, alkyl, hydroxyl, lower(C₁-C₃),alkoxy, amino, nitro, NH, N(C₁-C₃) straight chained or branched alkyl,O, S and N(C₁-C₄) straight chain or branched alkoxy; and,pharmaceutically acceptable salts thereof.
 36. A method according toclaim 35, wherein the non-antibiotic tetracycline compound selected fromthe group consisting of structures S-Z has the following provisos: wheneither R7 and R9 are hydrogen then R8 must be halogen; and when R6-a,R6, R5 and R9 are all hydrogen and R7 is hydrogen, amino, nitro,halogen, dimethylamino or diethylamino, then R8 must be halogen; andwhen R6-a is methyl, R6 and R9 are both hydrogen, R5 is hydroxyl, and R7is hydrogen, amino, nitro, halogen or diethylamino, then R8 is halogen;and when R6-a is methyl, R6 is hydroxyl, R5, R7 and R9 are all hydrogen,then R8 must be halogen; and when R6-a, R6 and R5 are all hydrogen, R9is methylamino and R7 is dimethylamino, then R8 Pmust be halogen; andwhen R6-a is methyl, R6 is hydrogen, R5 is hydroxyl, R9 is methylaminoand R7 is dimethylamino, then R8 must be halogen; and when R6-a ismethyl, R6, R5 and R9 are all hydrogen and R7 is cyano, then R8 must behalogen.
 37. A method according to claim 1, wherein said tetracyclinecompound has a photoirritancy factor of less than the photoirritancyfactor of doxycycline.
 38. A method according to claim 1, wherein saidtetracycline compound has a photoirritancy factor from about one toabout two.
 39. A method according to claim 38, wherein said tetracyclinecompound has a general formula:

wherein R7, R8, and R9 taken together are, respectively, hydrogen,hydrogen and dimethylamino.
 40. A method according to claim 1, whereinsaid tetracycline compound has a photoirritancy factor from about 1.0 toabout 1.2.
 41. A method according to claim 41, wherein said tetracyclinecompound is selected from the group consisting of:

wherein R7, R8, and R9 taken together in each case, have the followingmeanings: R7 R8 R9 hydrogen hydrogen amino hydrogen hydrogen palmitamideand

Structure L

Structure M

Structure N

Structure O

wherein R7, R8, and R9 taken together in each case, have the followingmeanings: R7 R8 R9 hydrogen hydrogen acetamido hydrogen hydrogendimethylaminoacetamido hydrogen hydrogen nitro hydrogen hydrogen aminoand

Structure P

wherein R8, and R9 taken together are, respectively, hydrogen and nitro.42. A method according to claim 1 wherein said treatment comprisesadministering a non-antibiotic tetracycline derivative topically.
 43. Amethod according to claim 42 wherein said non-antibiotic tetracyclinederivative is administered in a mouthwash.